Humanized anti-CCR2 antibodies and methods of use therefor

ABSTRACT

The present invention relates to a humanized antibody or functional fragment thereof which binds to a mammalian (e.g., human) CC-chemokine receptor 2 (CCR2) or a portion of the receptor and blocks binding of a ligand to the receptor. The invention further relates to a method of inhibiting the interaction of a cell bearing mammalian CCR2 with a ligand thereof, and to use of the antibodies and fragments in therapeutic, prophylactic and diagnostic methods.

RELATED APPLICATIONS

[0001] This application is a divisional of U.S. application Ser. No.09/497,625, filed Feb. 3, 2000, which is a continuation-in-part of U.S.application Ser. No. 09/359,193, filed Jul. 22, 1999, which is acontinuation-in-part of U.S. application Ser. No. 09/121,781, filed Jul.23, 1998, the entire teachings of all of which are incorporated hereinby reference.

BACKGROUND OF THE INVENTION

[0002] Over the past several years a growing family of leukocytechemoattractant/activating factors, termed chemokines, has beendescribed (Oppenheim, J. J. et al., Annu. Rev. Immunol., 9:617-648(1991); Schall and Bacon, Curr. Opin. Immunol., 6:865-873 (1994);Baggiolini, M., et al., Adv. Imunol., 55:97-179 (1994)). Members of thisfamily are produced and secreted by many cell types in response to earlyinflammatory mediators such as IL-1β or TNFα. The chemokine superfamilycomprises two main branches: the α-chemokines (or CXC chemokines) andthe β-chemokines (CC chemokines). The α-chemokine branch includesproteins such as IL-8, neutrophil activating peptide-2 (NAP-2), melanomagrowth stimulatory activity (MGSA/gro or GROα), and ENA-78, each ofwhich have attracting and activating effects predominantly onneutrophils. The members of the β-chemokine branch affect other celltypes such as monocytes, lymphocytes, basophils, and eosinophils(Oppenheim, J. J. et al., Annu. Rev. Immunol., 9:617-648 (1991);Baggiolini, M., et al., Adv. Imunol., 55:97-179 (1994); Miller andKrangel, Crit. Rev. Immunol., 12:17-46 (1992); Jose, P. J., et al., J.Exp. Med., 179:881-118 (1994); Ponath, P. D., et al., J. Clin. Invest.,97:604-612 (1996)), and include proteins such as monocyte chemotacticproteins 1-4 (MCP-1, MCP-2, MCP-3, and MCP-4), RANTES, and macrophageinflammatory proteins (MIP-1α, MIP-1β). Recently a new class ofmembrane-bound chemokines designated CX3C chemokines has been identified(Bazan, J. F., et al., Nature 385:640-644 (1997)). Chemokines canmediate a range of pro-inflammatory effects on leukocytes, such astriggering of chemotaxis, degranulation, synthesis of lipid mediators,and integrin activation (Oppenheim, J. J. et al., Annu. Rev. Immunol.,9:617-648 (1991); Baggiolini, M., et al., Adv. Imunol., 55:97-179(1994); Miller, M. D. and Krangel, M. S., Crit. Rev. Immunol., 12:17-46(1992)). Lately, certain β-chemokines have been shown to suppress HIV-1infection of human T cell lines in vitro (Cocchi, F., et al., Science(Wash. DC), 270:1811-1815 (1995)).

[0003] Chemokines bind to 7 transmembrane spanning (7TMS) Gprotein-coupled receptors (Murphy, P. M., Annu. Rev. Immunol.,12:593-633 (1994)). Some known receptors for the CC or β chemokinesinclude CCR1, which binds MIP-1α and RANTES (Neote, K., et al., Cell,72:415-425 (1993); Gao, J. L., J. Exp. Med., 1 77:1421-1427 (1993));CCR2, which binds chemokines including MCP-1, MCP-2, MCP-3 and MCP-4(Charo, I. F., et al., Proc. Natl. Acad. Sci. USA, 91:2752-2756 (1994);Myers, S. J., et al., J. Biol. Chem., 270:5786-5792 (1995); Gong et al.,J. Biol Chem 272:11682-11685 (1997); Garcia-Zepeda et al., J. Immunol.157:5613-5626 (1996)); CCR3, which binds chemokines including eotaxin,RANTES and MCP-3 (Ponath, P. D., et al., J. Exp. Med., 183:2437-2448(1996)); CCR4, which has been found to signal in response to MCP-1,MIP-1α, and RANTES (Power, C. A., et al., J. Biol. Chem.,270:19495-19500 (1995)); and CCR5, which has been shown to signal inresponse to MIP-1α, MIP-1β and RANTES (Boring, L., et al., J. Biol.Chem., 271 (13):7551-7558 (1996); Raport, C. J., J. Biol. Chem.,271:17161-17166 (1996); and Samson, M. et al., Biochemistry,35:3362-3367 (1996)).

[0004] CCR2 is expressed on the surface of several leukocyte subsets,and appears to be expressed in two slightly different forms (CCR2a andCCR2b) due to alternative splicing of the mRNA encoding thecarboxy-terminal region (Charo et al., Proc. Natl. Acad. Sci. USA91:2752-2756 (1994)). MCP-1 acts upon monocytes, lymphocytes andbasophils, inducing chemotaxis, granule release, respiratory burst andhistamine and cytokine release. Studies have suggested that MCP-1 isimplicated in the pathology of diseases such as rheumatoid arthritis,atherosclerosis, granulomatous diseases and multiple sclerosis (Koch, J.Clin. Invest. 90:772-79 (1992); Hosaka et al., Clin. Exp. Immunol.97:451-457(1994); Schwartz et al., Am. J. Cardiol. 71(6):9B-14B (1993);Schimmer et al., J. Immunol. 160:1466-1471 (1998); Flory et al., Lab.Invest. 69:396-404 (1993); Gong et al., J. Exp. Med. 186:131-137(1997)). Additionally, CCR2 can act as a co-receptor for HIV (Connor etal., J. Exp. Med. 185:621-628 (1997)). Thus, CCR2 receptor antagonistsmay represent a new class of important therapeutic agents.

SUMMARY OF THE INVENTION

[0005] The present invention relates to an antibody (immunoglobulin) orfunctional fragment thereof (e.g., an antigen-binding fragment) whichbinds to a mammalian CC-chemokine receptor 2 (also referred to as CCR2,CKR-2, MCP-1RA or MCP-1RB) or portion of the receptor (anti-CCR2). Inone embodiment, the antibody of the present invention or fragmentthereof has specificity for human or rhesus CCR2 or a portion thereof.In another embodiment, the antibody or fragment of the invention blocksbinding of a ligand (e.g., MCP-1, MCP-2, MCP-3, MCP-4) to the receptorand inhibits function associated with binding of the ligand to thereceptor (e.g., leukocyte trafficking). For example, as describedherein, antibodies and fragments thereof of the present invention whichbind human or rhesus CCR2 or a portion thereof, can block binding of achemokine (e.g., MCP-1, MCP-2, MCP-3, MCP-4) to the receptor and inhibitfunction associated with binding of the chemokine to the receptor. Inone embodiment, the antibody is monoclonal antibody (mAb) LS132.1D9(1D9) or an antibody which can compete with 1D9 for binding to humanCCR2 or a portion of human CCR2. Functional fragments of the foregoingantibodies are also envisioned.

[0006] In another embodiment, the antibody or functional fragment of thepresent invention binds human CCR2 or a portion thereof, and inhibitshuman immunodeficiency virus (HIV) binding to the receptor, therebyinhibiting function associated with binding of HIV to the receptor(e.g., HIV antigen release and infectivity). In one embodiment, theantibody is monoclonal antibody 1D9 or an antibody which can competewith 1D9 for binding to human CCR2 or a portion of human CCR2.

[0007] The present invention also relates to an antibody or functionalfragment thereof (e.g., an antigen-binding fragment) which binds to amammalian CCR2 or portion of the receptor and provides increasedfluorescent staining intensity of CCR2 or compositions comprising CCR2relative to other anti-CCR2 antibodies. In one embodiment, the antibodyis monoclonal antibody 1D9 or LS 132.8G2 (8G2) or an antibody which cancompete with 1D9 or 8G2 for binding to human CCR2 or a portion of humanCCR2.

[0008] The present invention also relates to a humanized immunoglobulinor antigen-biding fragment thereof having binding specificity for CCR2,said immunoglobulin comprising an antigen binding region of nonhumanorigin (e.g., rodent) and at least a portion of an immunoglobulin ofhuman origin (e.g., a human framework region, a human constant region ofthe gamma type). In one embodiment, the humanized immunoglobulin orfragment thereof described herein can compete with 1D9 for binding toCCR2. In a preferred embodiment, the antigen binding region of thehumanized immunoglobulin is derived from monoclonal antibody 1D9 (e.g.,an immunoglobulin comprising the variable regions of the light and heavychains as shown in FIG. 7 (SEQ ID NO: 9) and FIG. 8 (SEQ ID NO: 10),respectively).

[0009] For example, the humanized immunoglobulin or antigen-bindingfragment thereof can comprise an antigen binding region comprising atleast one complementarity determining region (CDR) of nonhuman origin,and a framework region (FR) derived from a human framework region. Inone aspect, the humanized immunoglobulin having binding specificity forCCR2 comprises a light chain comprising at least one CDR derived from anantibody of nonhuman origin which binds CCR2 and a FR derived from alight chain of human origin (e.g., from HF-21/28), and a heavy chaincomprising a CDR derived from an antibody of nonhuman origin which bindsCCR2 and a FR derived from a heavy chain of human origin (e.g., from4B4′CL). In another aspect, the light chain comprises three CDRs derivedfrom the light chain of the 1D9 antibody, and the heavy chain comprisesthree CDRs derived from the heavy chain of the 1D9 antibody.

[0010] The present invention also relates to humanized immunoglobulinlight chains and antigen-binding fragments thereof (e.g., comprisingCDR1, CDR2 and CDR3 of the light chain of the 1D9 antibody, and a humanlight chain FR), and to humanized immunoglobulin heavy chains andantigen-binding fragments thereof (e.g., comprising CDR1, CDR2 and CDR3of the heavy chain of the 1D9 antibody, and a human heavy chain FR). Ina preferred embodiment, the invention relates to humanized heavy andlight chains described herein (e.g., a humanized light chain comprisingthe variable region of the light chain shown in FIG. 7 (SEQ ID NO: 9), ahumanized heavy chain comprising the variable region of the heavy chainshown in FIG. 8 (SEQ ID NO: 10). Also encompassed are humanizedimmunoglobulins comprising one or more humanized light and/or heavychains.

[0011] The invention further relates to isolated nucleic acid moleculescomprising a nucleic acid sequence which encodes a humanizedimmunoglobulin of the present invention (e.g., a single chain antibody),as well as to isolated nucleic acid molecules comprising a sequencewhich encodes a humanized immunoglobulin light chain (e.g., comprisingnucleotides 52-390 of SEQ ID NO: 95) or heavy chain (e.g., comprisingnucleotides 58-411 of SEQ ID NO: 96) of the present invention. Forexample, the present invention provides a gene (e.g., a fused gene)encoding a humanized immunoglobulin light or heavy chain comprising afirst nucleic acid sequence encoding an antigen binding region derivedfrom murine 1D9 monoclonal antibody; and a second nucleic acid sequenceencoding at least a portion of a constant region of an immunoglobulin ofhuman origin.

[0012] The present invention further relates to a construct comprising anucleic acid molecule encoding a humanized immunoglobulin having bindingspecificity for CCR2 or a chain of such an immunoglobulin. For example,an expression vector comprising a gene (e.g., a fused gene) encoding ahumanized immunoglobulin light chain, comprising a nucleotide sequenceencoding a CDR derived from a light chain of a nonhuman antibody havingbinding specificity for CCR2, and a framework region derived from alight chain of human origin, is provided. An expression vectorcomprising a gene encoding a humanized immunoglobulin heavy chain,comprising a nucleotide sequence encoding a CDR derived from a heavychain of a nonhuman antibody having binding specificity for CCR2, and aframework region derived from a heavy chain of human origin is anotherexample of such a construct.

[0013] The present invention also relates to a host cell comprising anucleic acid molecule of the present invention, including one or moreconstructs comprising a nucleic acid molecule of the present invention.In one embodiment, the invention relates to a host cell comprising afirst recombinant nucleic acid encoding a humanized immunoglobulin lightchain, and a second recombinant nucleic acid encoding a humanizedimmunoglobulin heavy chain, said first nucleic acid comprising anucleotide sequence encoding a CDR derived from the light chain ofmurine 1D9 antibody and a framework region derived from a light chain ofhuman origin; and said second nucleic acid comprising a nucleotidesequence encoding a CDR derived from the heavy chain of murine 1D9antibody and a framework region derived from a heavy chain of humanorigin.

[0014] The present invention also provides a method of preparing ahumanized immunoglobulin comprising maintaining a host cell of thepresent invention under conditions appropriate for expression of ahumanized immunoglobulin, whereby a humanized immunoglobulin chain(s) isexpressed and a humanized immunoglobulin is produced. The method canfurther comprise the step of isolating the humanized immunoglobulin.

[0015] The humanized immunoglobulins of the present invention can beless immunogenic than their murine or other nonhuman counterparts. Thus,the humanized immunoglobulins described herein can be used astherapeutic agents in humans, for example to control lymphocyte homingto mucosal lymphoid tissue, thereby, reducing inflammatory responses.

[0016] The invention further relates to a humanized immunoglobulin ofthe present invention for use in diagnosis or therapy (includingprophylaxis). In one embodiment, the invention relates to a humanizedimmunoglobulin of the present invention for use in the treatment ofdiseases associated with leukocyte infiltration of tissues, for example,in the treatment of inflammatory diseases, autoimmune diseases, graftrejection, HIV infection and monocyte-mediated disorders such asatherosclerosis.

[0017] In another aspect, the invention relates to use of a humanizedimmunoglobulin of the present invention for the manufacture of amedicament for the treatment of diseases associated with leukocyteinfiltration of tissues, for example, in the treatment of inflammatorydiseases, autoimmune diseases, monocyte-mediated disorders such asatherosclerosis, graft rejection, or HIV infection.

[0018] The present invention further relates to a method of inhibitingthe interaction of a cell bearing mammalian (e.g., human, non-humanprimate or murine) CCR2 with a ligand thereof, comprising contacting thecell with an effective amount of an antibody or functional fragmentthereof which binds to a mammalian CCR2 or a portion of CCR2. Suitablecells include granulocytes, leukocytes, such as monocytes, macrophages,basophils and eosinophils, mast cells, and lymphocytes including T cells(e.g., CD8+ cells, CD4+ cells, CD25+ cells, CD45RO+ cells), and othercells expressing CCR2 such as a recombinant cell expressing CCR2 (e.g.,transfected cells). In a particular embodiment, the antibody is 1D9 oran antibody which can compete with 1D9 for binding to human CCR2 or aportion of human CCR2.

[0019] Another embodiment of the invention relates to a method ofinhibiting the interaction of a cell bearing mammalian CCR2 with achemokine, comprising contacting said cell with an effective amount ofan antibody or functional fragment thereof which binds to CCR2 or aportion of said receptor. In one embodiment of the method, the antibodyor functional fragment thereof is any one or more of 1D9, anantigen-binding fragment of 1D9 or an antibody or fragment thereofhaving an epitopic specificity which is the same as or similar to thatof 1D9. Furthermore, the invention relates to a method of inhibiting afunction associated with binding of a chemokine to CCR2, comprisingadministering an effective amount of an antibody or functional fragmentthereof which binds to a mammalian CCR2 protein or a portion of saidreceptor. In one aspect of the method, the antibody or functionalfragment thereof is any one or more of 1D9, an antigen-binding fragmentof 1D9 or an antibody or fragment thereof having an epitopic specificitywhich is the same as or similar to that of 1D9.

[0020] Another aspect of the invention is a method of identifyingexpression of a mammalian CCR2 or portion of the receptor by a cell.According to the method, a composition comprising a cell or fractionthereof (e.g., a membrane fraction) is contacted with an antibody orfunctional fragment thereof (e.g., 1D9 or 8G2) which binds to amammalian CCR2 protein or portion of the receptor under conditionsappropriate for binding of the antibody thereto, and the formation of acomplex between said antibody or fragment and said protein or portionthereof is detected. Detection of the complex, directly or indirectly,indicates the presence of the receptor on the cell. The presentinvention also relates to a kit for use in detecting the presence ofCCR2 or a portion thereof in a biological sample, comprising an antibodyor functional fragment thereof which binds to a mammalian CC-chemokinereceptor 2 or a portion of said receptor, and one or more ancillaryreagents suitable for detecting the presence of a complex between saidantibody or fragment and said protein or portion thereof.

[0021] Also encompassed by the present invention are methods ofidentifying additional ligands or other substances which bind amammalian CCR2 protein, including inhibitors and/or promoters ofmammalian CCR2 function. For example, agents having the same or asimilar binding specificity as that of an antibody of the presentinvention or functional fragment thereof can be identified by acompetition assay with said antibody or fragment. Thus, the presentinvention also encompasses methods of identifying ligands or othersubstances which bind the CCR2 receptor, including inhibitors (e.g.,antagonists) or promoters (e.g., agonists) of receptor function. In oneembodiment, cells which naturally express CCR2 receptor protein orsuitable host cells which have been engineered to express a CCR2receptor or variant encoded by a nucleic acid introduced into said cellsare used in an assay to identify and assess the efficacy of ligands,inhibitors or promoters of receptor function. Such cells are also usefulin assessing the function of the expressed receptor protein orpolypeptide.

[0022] Thus, the invention also relates to a method of detecting oridentifying an agent which binds a mammalian CCR2 or ligand bindingvariant thereof, comprising combining an agent to be tested, an antibodyor antigen-binding fragment of the present invention (e.g., monoclonalantibody 1D9, an antibody having an epitopic specificity which is thesame as or similar to that of 1D9, antigen-binding fragments of 1D9,monoclonal antibody 8G2, an antibody having an epitopic specificitywhich is the same as or similar to that of 8G2, and antigen-bindingfragments of 8G2) and a composition comprising a mammalian CCR2 proteinor a ligand binding variant thereof. The foregoing components can becombined under conditions suitable for binding of the antibody orantigen-binding fragment to mammalian CCR2 protein or a ligand bindingvariant thereof, and binding of the antibody or fragment to themammalian CCR2 protein or ligand binding variant is detected ormeasured, either directly or indirectly, according to methods describedherein or other suitable methods. A decrease in the amount of complexformed relative to a suitable control (e.g., in the absence of the agentto be tested) is indicative that the agent binds said receptor orvariant. The composition comprising a mammalian CCR2 protein or a ligandbinding variant thereof can be a membrane fraction of a cell bearingrecombinant CCR2 protein or ligand binding variant thereof. The antibodyor fragment thereof can be labeled with a label such as a radioisotope,spin label, antigen label, enzyme label, fluorescent group andchemiluminescent group. These and similar assays can be used to detectagents, including ligands (e.g., chemokines which interact with CCR2) orother substances, including inhibitors or promoters of receptorfunction, which can bind CCR2 and compete with the antibodies describedherein for binding to the receptor.

[0023] According to the present invention, ligands, inhibitors orpromoters of receptor function can be identified in a suitable assay,and further assessed for therapeutic effect. Inhibitors of receptorfunction can be used to inhibit (reduce or prevent) receptor activity,and ligands and/or promoters can be used to induce (trigger or enhance)normal receptor function where indicated. The present invention alsoprovides a method of treating inflammatory diseases, autoimmunediseases, atherosclerosis, and graft rejection, or HIV infection,comprising administering an inhibitor of receptor function (e.g.,chemokine binding or HIV binding) to an individual (e.g., a mammal, suchas a human). The present invention further provides a method ofstimulating receptor function by administering a novel ligand orpromoter to an individual, providing a new approach to selectivestimulation of leukocyte function, which is useful, for example, in thetreatment of infectious diseases and cancer.

[0024] Another aspect of the invention relates to a method of inhibitingHIV infection of a cell which expresses a mammalian CCR2 or portionthereof, comprising contacting the cell with an effective amount of anantibody or functional fragment thereof which binds to a mammalian CCR2or portion of the receptor and inhibits HIV binding and infection. In aparticular embodiment of the invention, the antibody or functionalfragment thereof is any of 1D9, an antibody having an epitopicspecificity which is the same as or similar to that of 1D9, an antibodywhich can compete with 1D9 for binding to human CCR2, andantigen-binding fragments thereof.

[0025] Also encompassed by the present invention is a method ofinhibiting (e.g., treating) HIV in a patient, comprising administeringto the patient an effective amount of an antibody or functional fragmentthereof which binds to a mammalian CCR2 or a portion of said receptorand inhibits HIV binding to the CCR2 receptor. The anti-CCR2 antibody orfragment can be administered alone or in combination with one or moreadditional therapeutic agents, e.g., one or more antibodies which bind aco-receptor for HIV infection and inhibit binding to said co-receptor,such as an anti-CCR3, anti-CCR5, and/or anti-CXCR4 antibody.

[0026] Another aspect of the invention also relates to a method ofpreventing or inhibiting HIV infection in an individual, comprisingadministering to the individual an effective amount of an antibody orfunctional fragment thereof which binds to CCR2 and inhibits HIV bindingto CCR2. According to the method, preventing HIV infection includestreatment in order to prevent (reduce or eliminate) infection of newcells in an infected individual or in order to prevent infection in anindividual who may be, may have been or has been exposed to HIV. Forexample, individuals such as an HIV infected individual, a fetus of anHIV infected female, or a health care worker can be treated according tothe method of the present invention.

[0027] The present invention also encompasses a method of inhibitingleukocyte trafficking in a patient, comprising administering to thepatient an effective amount of an antibody or functional fragmentthereof which binds to a mammalian CCR2 or portion of said receptor andinhibits function associated with binding of a ligand to the receptor.

[0028] The present invention also relates to a method of inhibiting ortreating CCR2-mediated disorders, such as inflammatory disorders,comprising administering to a patient an effective amount of an antibodyor functional fragment thereof which binds to a mammalian CCR2 orportion of said receptor and inhibits CCR2-mediated function. Forexample, the invention relates to a method of inhibiting or treatingstenosis or restenosis of the vasculature comprising administering to apatient an effective amount of an antibody or functional fragmentthereof which binds to a mammalian CCR2 or portion of said receptor andinhibits CCR2-mediated function.

[0029] The present invention further relates to an antibody or fragmentthereof as described herein (e.g., monoclonal antibody 1D9 or anantigen-binding fragment thereof) for use in therapy (includingprophylaxis) or diagnosis, and to the use of such an antibody orfragment for the manufacture of a medicament for the treatment of aCCR2-mediated disorder, or other disease or inflammatory condition asdescribed herein.

BRIEF DESCRIPTION OF THE FIGURES

[0030] FIGS. 1A-1O are fluorescence activated cell scanning (FACS)histogram profiles illustrating that mAbs 1D9 and 8G2 stain CCR2transfectants but not CCR5 or CCR1 transfectants. L1/2 (also referred toherein as L1.2) murine pre-B lymphoma host cells were transfected withCCR2, CCR5 and CCR1 as indicated, and stained with antibodies withdifferent receptor specificities. Staining was analyzed by flowcytometry.

[0031] FIGS. 2A-2L are FACS dot plots showing expression of CCR2 on mostmonocytes, a subpopulation of lymphocytes and a small subset ofgranulocytes. Whole blood cells were stained with one of three anti-CCR2mAbs (5A11, generated using a peptide consisting of the first 32 aminoacids of the CCR2 amino-terminus as an immunogen, and 1D9 and 8G2generated as described herein using CCR2b L1/2 cell transfectants as theimmunogen). Staining was analyzed by flow cytometry, and the lymphocyte,granulocyte and monocyte populations were gated using the forward andside light scatter. The X-axis represents forward light scatter (ameasure of cell size), and the Y-axis fluorescence intensity of stainingfor CCR2. The level of negative control staining is indicated by a line.

[0032] FIGS. 3A-3I are FACS dot plots showing that mAb 1D9 stains an IgEpositive population in peripheral blood (basophils) using two-colorstaining for IgE and CCR2. Whole blood cells were first stained witheither a negative control antibody (anti-Flag), anti-CCR2 antibody 1D9,or an anti-CXCR1 antibody, as indicated, and detected by ananti-mouse-FITC conjugate. A second staining was done using either PBSor a biotinylated antibody specific for IgE or CD16, as indicated, anddetected with a streptavidin-phycoerythrin. Staining was analyzed byflow cytometry.

[0033]FIG. 4 illustrates that mAb 1D9 inhibits [¹²⁵I]MCP-1 binding toTHP-1 cell membranes. 3.0 μg of THP-1 membrane protein was incubatedwith 0.1 nM [¹²⁵I]MCP-1 in the presence of various concentrations of 1D9or the isotype-matched anti-CXCR3 antibody 1 C6. The amount of boundtracer was determined by separation of free from bound by filtration andscintillation counting. The data was analyzed to determine the IC₅₀value by non-linear regression using a 4-parameter logistic equationwith KaleidaGraph software.

[0034]FIG. 5 illustrates that mAb 1D9 inhibits [¹²⁵I ]MCP-1 binding tofresh human PBMC. Freshly isolated peripheral blood mononuclear cells(500,000) were incubated with 0.1 nM [¹²⁵I ]MCP-1 in the presence ofvarious concentrations of 1D9 or the isotype-matched anti-CXCR3 antibody1 C6. The amount of bound tracer was determined by separation of freefrom bound by filtration and scintillation counting. The data wasanalyzed to determine the lC₅₀ value as for FIG. 4.

[0035]FIGS. 6A and 6B are graphs demonstrating that mAb 1D9 inhibitsMCP-1-induced chemotaxis, but not RANTES-induced chemotaxis, of freshPBMC. FIG. 6A shows the results of chemotaxis assays of PBMC to 10 nMMCP-1 with no antibody, or 0.1 or 10 μg/ml of 1D9 or nonspecific murineIgG2a. The spontaneous nonspecific migration is also indicated. FIG. 6Bshows the results of chemotaxis assays of PBMC to 10 nM RANTES with noantibody, 10 μg/ml 1D9 or 10 μg/ml nonspecific murine IgG2a. Thespontaneous nonspecific migration in the absence of RANTES is alsoindicated.

[0036]FIG. 7 shows the amino acid sequence (SEQ ID NO: 9) of the kappalight chain variable region of the murine 1D9 antibody. The CDRs arehighlighted in bold.

[0037]FIG. 8 shows the amino acid sequence (SEQ ID NO: 10) of the heavychain variable region of the murine 1D9 antibody. The CDRs arehighlighed in bold.

[0038]FIG. 9 illustrates the canonical classes of CDRs in the murine 1D9V_(K) region. “Chothia Canonical Classes” indicates where the canonicalclasses as defined by Chothia and his colleagues (Chothia and Lesk, J.Mol. Biol. 197:901 (1987); Chothia et al., Nature 34:877 (1989);Tramontano et al., J. Mol. Biol. 215:175 (1990); and Chothia et al., J.Mol. Biol. 227:799 (1992)) were used, while “Martin Canonical Classes”signifies where the canonical classes defined by Martin and Thornton(Martin and Thornton, J. Mol. Biol. 263:800 (1996)) were used. FRresidues are highlighted in bold.

[0039]FIG. 10 illustrates the canonical classes of CDRs in the murine1D9 V_(H) region. “Chothia Canonical Classes” indicates where thecanonical classes as defined by Chothia and his colleagues (Chothia andLesk, J. Mol. Biol. 197:901 (1987); Chothia et al., Nature 34:877(1989); Tramontano et al., J. Mol. Biol. 215:175 (1990); and Chothia etal., J. Mol. Biol. 227:799 (1992)) were used, while “Martin CanonicalClasses” signifies where the canonical classes defined by Martin andThornton (Martin and Thornton, J. Mol. Biol. 263:800 (1996)) were used.FR residues are highlighted in bold.

[0040]FIG. 11 shows the amino acid sequences of various versions of thehumanised 1D9 V_(K) region (SEQ ID NOS: 12-15, respectively). Where the1D9 V_(K) region residues (SEQ ID NO: 9) and the human HF-21/28 V_(K)region (SEQ ID NO: 11) sequences match a dot [.] is shown. Where noamino acid is present at a specific residue position a dash [-] isshown. Where an amino acid in the HF-21/28 FRs is changed in thehumanised 1D9 VK region, it is highlighted in bold. The CDRs aredescribed by the use of nomenclature [==L1==]. The numbering used isaccording to Kabat et al., Sequences of proteins of immunologicalinterest, Fifth edition, U.S. Department of Health and Human Services,U.S. Government Printing Office (1991).

[0041]FIG. 12 shows the amino acid sequences of various versions of thehumanised 1D9 V_(H) region (SEQ ID NOS: 17-20, respectively). Where the1D9 V_(H) region residues (SEQ ID NO: 10) and the human 4B4′CL V_(H)region sequences (SEQ ID NO: 16) match a dot [.] is shown. Where noamino acid is present at a specific residue position a dash [-] isshown. Where an amino acid in the 4B4′CL is changed in the humanised 1D9VH region, it is highlighted in bold. The CDRs are described by the useof nomenclature [==H1==], while [---] denotes part of the H1 structureloop. The numbering used is according to Kabat et al., Sequences ofproteins of immunological interest, Fifth edition, U.S. Department ofHealth and Human Services, U.S. Government Printing Office (1991).

[0042]FIG. 13 shows a comparison of a portion of the murine 1D9 V_(K)region (SEQ ID NO: 21) with mouse germline VK gene sequences (SEQ IDNOS: 22-33, respectively). “Identical residues” represents the number ofidentical residues in a mouse germline V_(K) region to the murine 1D9V_(K) region. Where the 1D9 V_(K) region sequence and the mouse germlineV_(K) region sequences match a dot [.] is shown. Where no amino acid ispresent at a specific residue position a dash [-] is shown.

[0043]FIG. 14 shows a comparison of a portion of the murine 1D9 V_(H)region (SEQ ID NO: 34) with mouse germline V_(H) gene sequences (SEQ IDNOS: 35-53, respectively). “Identical residues” represents the number ofidentical residues in a mouse germline V_(H) region to the murine 1D9V_(H) region. Where the 1D9 V_(H) region sequence and the mouse germlineV_(H) region sequences match a dot [.] is shown. Where no amino acid ispresent at a specific residue position a dash [-] is shown.

[0044]FIG. 15 shows a comparison of the murine 1D9 V_(K) region (SEQ IDNO: 9) with the most homologous seventeen human V_(K) amino acidsequences (SEQ ID NOS: 54-70, respectively). “ID” represents thepercentage identity of the human V_(K) sequences to the murine 1D9 V_(K)region. Where the 1D9 V_(K) region residues and the human V_(K) regionsequences match a dot [.] is shown. Where no amino acid is present at aspecific residue position a dash [-] is shown. “S” indicates amino acidpositions on the surface of the F_(V) domain. “C” indicates residueslocated within the core of the F_(V) domain. Residues within 5 Å of aCDR are defined using capital letters, while those located further awayare described with a lower case letter. The CDRs themselves aredescribed by the use of the nomenclature ==L1==. “v” denotes the Vernierresidues (Foote and Winter, J. Mol. Biol. 224:487 (1992)) located in theFRs. Those residues in the human V_(K) region sequences which areunderlined differ from their closest human V_(K) germline gene. Thenumbering used is as according to Kabat et al., Sequences of proteins ofimmunological interest, Fifth edition, U.S. Department of Health andHuman Services, U.S. Government Printing Office (1991).

[0045]FIG. 16 shows a comparison of the murine 1D9 V_(K) region with themost homologous seventeen human V_(K) amino acid sequences. “1D”indicates the percentage identity of the human V_(K) sequence to themurine 1D9 V_(K) region. “Surface” indicates the number of identicalresidues on the surface. “Core” indicates the number of identicalresidues within the core of the F_(V) domain. “CDR” indicates the numberof identical residues within the CDRs. “FR” indicates the number ofidentical residues within the FRs. “FR Surface” indicates the number ofidentical residues which are surface exposed. “FR Core” indicates thenumber of identical residues which are located within the core of theF_(V) domain. “FR Near CDR” represents the number of identical residuesamongst the FR amino acids within 5 Å of a CDR. “Vernier” indicates thenumber of identical residues amongst the 14 Vernier amino acids (Footeand Winter, J. Mol. Biol. 224:487 (1992)). “V_(K)” indicates the numberof identical residues within the V_(K) gene. “J Chain” indicates thenumber of identical residues within the J chain gene. “L1 Len” to “L3Len” defines the number of residues in each CDR, while “L1 Class” to “L3Class” describes the canonical class of the CDR according to Martin andThornton (Martin and Thornton, J. Mol. Biol. 263:800 (1996)).

[0046] FIGS. 17A-17B show a comparison of the murine 1D9 V_(H) region(SEQ ID NO: 10) with the most homologous 24 human V_(H) amino acidsequences (SEQ ID NOS: 71-94, respectively). “ID” represents thepercentage identity of the human V_(H) sequences to the murine 1D9 V_(H)region. Where the 1D9 V_(H) region residues and the human V_(H) regionsequences match a dot [.] is shown. Where no amino acid is present at aspecific residue position a dash [-] is shown. “S” indicates amino acidpositions on the surface of the F_(V) domain. “C” indicates residueslocated within the core of the F_(V) domain. Residues within 5 Å of aCDR are defined using capital letters, while those located farther awayare described with a lower case letter. The CDRs themselves aredescribed by the use of the nomenclature ==H1==. “v” denotes the Vernierresidues (Foote and Winter, J. Mol. Biol. 224:487 (1992)) located in theFRs. Those residues in the human V_(H) region sequences which areunderlined differ from their closest human V_(H) germline gene. Thenumbering used is as according to Kabat et al., Sequences of proteins ofimmunological interest, Fifth edition, U.S. Department of Health andHuman Services, U.S. Government Printing Office (1991).

[0047] FIGS. 18A-18B show a comparison of the murine 1D9 V_(H) regionwith the most homologous 24 human V_(H) amino acid sequences. “ID”indicates percentage identity of the human V_(H) sequence to the murine1D9 V_(H) region. “All” indicates the number of identical residues inthe whole of the human V_(H) region when compared to the whole of themurine 1D9 V_(H) region. “Surface” indicates the number of identicalresidues on the surface. “Core” indicates the number of identicalresidues within the core of the F_(V) domain. “CDR” indicates the numberof identical residues within the CDRs. “FR” indicates the number ofidentical residues within the Frs. “FR Surface” indicates the number ofidentical residues which are surface exposed. “FR Core” indicates thenumber of identical residues which are located within the core of theF_(V) domain. “FR Near CDR” represents the number of identical residuesamongst the FR amino acids within 5 Å of a CDR. “Vernier” indicates thenumber of identical residues amongst the 14 Vernier amino acids (Footeand Winter, J. Mol. Biol. 224:487 (1992)). “V_(H)” indicates the numberof identical residues within the V_(H) gene. “J Chain” indicates thenumber of identical residues within the J chain gene. “H1 Size” to “H3Size” define the number of residues in each CDR, while “H1 Class” and“H2 Class” describe the canonical class of the CDR according to Martinand Thornton, (J. Mol. Biol. 263:800 (1996)).

[0048] FIGS. 19A-19C show the alignment of amino acid sequences leadingto the design of the first (1D9RK_(A)) and second (1D9RK_(B)) humanisedversions of the 1D9 antibody kappa light chain variable region. Aminoacids identical to the mouse 1D9 at a particular residue position incolumn 7 are not shown; “-” indicates no amino acid is located at thisresidue position. Boldface type indicates positions in FRs and CDRswhere the human amino acid residue was replaced by the correspondingmouse residue. “Δ” indicates the numbering of changes in the human FRsof 1D9RK_(A). “Mouse 1D9V_(K)” indicates the amino acid sequence of theV_(K) region from the murine 1D9 kappa light chain variable region.“Mouse κ-II” indicates the consensus sequence of mouse V_(K) regionsfrom Kabat subgroup κ-II. “Human κ-II” indicates the consensus sequenceof human V_(K) regions from Kabat subgroup κ-II. “HF-21/28” indicatesthe amino acid sequence of the light chain variable region from thehuman HF-21/28 antibody (Chastagner et al., Gene 10](2):305-6 (1991)).The number in parenthesis (005056) is the Kabat database ID number.“Surface or Core” indicates the position of the amino acid in relationto the rest of the residues in both chains of the antibody variableregions. Residues within 5 Å of a CDR are defined using capital letters.“1D⁹RK_(A)” indicates the amino acid sequence of the first version ofthe humanised 1D9 V_(K) region. “1D⁹RK_(B)” indicates the amino acidsequence of the second version of the humanised 1D9 V_(K) region.

[0049] FIGS. 20A-20C show the alignment of amino acid sequences leadingto the design of the first (1D⁹RH_(A)) and second (1D9RH_(B)) humanisedhuman versions of the 1D9 antibody kappa heavy chain variable region.Amino acids identical to the mouse 1D9 at a particular residue positionin column 7 are not shown. “-” indicates that no amino acid is locatedat this residue position. Boldface type indicates positions in the FRsand CDRs where the human amino acid residue was replaced by thecorresponding mouse residue. “Δ” indicates the numbering of changes inthe human FRs of 1D9RH_(A). “Mouse 1D9 V_(H)” indicates the amino acidsequence of the V_(H) region from the murine 1D9 heavy chain variableregion. “Mouse IIIc” indicates the consensus sequence of mouse V_(H)regions from Kabat subgroup IIIc. “Human III” indicates the consensussequence of human V_(H) regions from Kabat subgroup III. “4B4′CL”indicates the amino acid sequence of the heavy chain variable regionfrom the human 4B4′CL antibody (Sanz et al., Journal of Immunology142:883 (1989)). The number in parenthesis (000490) is the Kabatdatabase ID number. “Surface or Core” indicates the position of theamino acid in relation to the rest of the residues in both chains of theantibody variable regions. Residues within 5 Å of a CDR are definedusing capital letters. “1D9RH_(A)” indicates the amino acid sequence ofthe first version of the humanised 1D9 V_(H) region. “1D9RH_(B)”indicates the amino acid sequence of the second version of the humanised1D9 V_(H) region.

[0050]FIG. 21 shows the nucleotide sequence, complement and encodedamino acid sequence of the murine antibody 1D9 heavy chain variableregion. The leader sequence and a portion of the constant region arealso shown. The illustrated nucleotide sequence is SEQ ID NO: 96, thecomplementary sequence is SEQ ID NO: 99, and the amino acid sequence isSEQ ID NO: 100.

[0051]FIG. 22 shows the nucleotide sequence, complement and encodedamino acid sequence of the murine antibody 1D9 kappa light chainvariable region. The leader sequence and a portion of the constantregion are also shown. The illustrated nucleotide sequence is SEQ ID NO:95, the complementary sequence is SEQ ID NO: 101, and the amino acidsequence is SEQ ID NO: 102.

[0052]FIG. 23 shows the nucleotide sequence of the humanized heavy chain1D9RH_(A). The indicated enzyme sites were added for cloning into thevector pLKTOK38. The vector also has human leader and constant regions.The illustrated nucleotide sequence is SEQ ID NO: 97, the complementarysequence is SEQ ID NO: 103, and the amino acid sequence is SEQ ID NO:104.

[0053]FIG. 24 shows the nucleotide sequence of the humanized light chain1D⁹RK_(A). The indicated enzyme sites were added for cloning into thevector pLKTOK38. The vector also has human leader and constant regions.The bracketed Y indicates a residue which changes to aspartate when putinto the vector pLKTOK38. The illustrated nucleotide sequence is SEQ IDNO: 98, the complementary sequence is SEQ ID NO: 105, and the amino acidsequence is SEQ ID NO: 106.

DETAILED DESCRIPTION OF THE INVENTION

[0054] The present invention relates to an antibody (anti-CCR2) orfunctional fragment thereof which binds mammalian CC-chemokine receptor2 (CCR2, CKR-2, MCP-1RA or MCP-1RB) or a portion of CCR2. In oneembodiment, the antibody has specificity for human or rhesus CCR2 orportion thereof. In one embodiment, the antibodies (immunoglobulins) areraised against an isolated and/or recombinant mammalian CCR2 or portionthereof (e.g., peptide) or against a host cell which expresses mammalianCCR2. In a preferred embodiment, the antibodies specifically bind humanCCR2 receptor(s) (e.g., CCR2a and/or CCR2b) or a portion thereof, and ina particularly preferred embodiment the antibodies have specificity fora naturally occurring or endogenous human CCR2. As usedherein,“CC-chemokine receptor 2” (“CCR2”) refers to CC-chemokinereceptor 2a and/or CC-chemokine receptor 2b. Antibodies or functionalfragments thereof which can inhibit one or more functions characteristicof a mammalian CCR2, such as a binding activity (e.g., ligand, inhibitorand/or promoter binding), a signaling activity (e.g., activation of amammalian G protein, induction of a rapid and transient increase in theconcentration of cytosolic free calcium [Ca²⁺]i), and/or stimulation ofa cellular response (e.g., stimulation of chemotaxis, exocytosis orinflammatory mediator release by leukocytes, integrin activation) arealso encompassed by the present invention, such as an antibody which caninhibit binding of a ligand (i.e., one or more ligands) to CCR2 and/orone or more functions mediated by CCR2 in response to a ligand. Forexample, in one aspect, the antibodies or functional fragments thereofcan inhibit (reduce or prevent) the interaction of receptor with anatural ligand, such as MCP-1, MCP-2, MCP-3 and/or MCP-4. In anotheraspect, an antibody or functional fragment thereof that binds to CCR2can inhibit binding of MCP-1, MCP-2, MCP-3 and/or MCP-4 and/or HIV tomammalian CCR2 (e.g., human CCR2, non-human primate CCR2, murine CCR2).The antibodies or functional fragments thereof of the present inventioncan inhibit functions mediated by human CCR2, including leukocytetrafficking, HIV entry into a cell, T cell activation, inflammatorymediator release and/or leukocyte degranulation. Preferably, theantibodies or fragments can bind CCR2 with an affinity of at least about0.1×10⁻⁹ M, preferably at least about 1×10⁻⁹ M, and more preferably atleast about 3×10⁻⁹ M. In a particular embodiment, antibodies orfunctional fragments thereof demonstrate inhibition of chemokine-induced(e.g., MCP-1-induced) chemotaxis of cells (e.g., PBMC) at less thanabout 150 μg/ml, preferably less than about 100 μg/ml, more preferablyless than about 50 μg/ml, and even more preferably less than about 20μg/ml.

[0055] In a further embodiment of the invention, the antibodies orfunctional fragments thereof of the invention can inhibit binding of aCCR2 ligand (e.g., a chemokine) to CCR2 with an IC₅₀ of less than about1.0 μg/ml, preferably less than about 0.05 μg/ml, and more preferablyless than about 0.005 μg/ml.

[0056] Murine monoclonal antibodies specific for CCR2, designated 1D9and 8G2, were produced as described herein. In a preferred embodiment,the antibodies of the present invention bind human CCR2, and have anepitopic specificity which is the same as or similar to that of murine1D9 or 8G2 antibody described herein. Antibodies with an epitopicspecificity which is the same as or similar to that of murine 1D9monoclonal antibody can be identified by their ability to compete withmurine 1D9 monoclonal antibody for binding to human CCR2 (e.g., to cellsbearing human CCR2, such as transfectants bearing CCR2, CD8+ cells, CD4+cells, CDR45RO+ cells, CD25+ cells, monocytes, dendritic cells,macrophages and basophils). Similarly, antibodies with an epitopicspecificity which is the same as or similar to that of murine 8G2monoclonal antibody can be identified by their ability to compete withmurine 8G2 monoclonal antibody for binding to human CCR2. Using receptorchimeras (Rucker et al., Cell 87:437-446 (1996)), the binding site ofmAbs 1D9 and 8G2 has been mapped to the amino-terminal domain of humanCC-chemokine receptor 2, specifically to an epitope comprising fromabout amino acid 1 to about amino acid 30 of the protein. Using these orother suitable techniques, antibodies having an epitopic specificitywhich is the same as or similar to that of an antibody of the presentinvention can be identified. mAbs 1D9 and 8G2 have epitopic specificityfor the amino-terminal domain of the CCR2 receptor, e.g., from aboutamino acid number 1 to about amino acid number 30 of the receptorprotein. Thus, the invention pertains to an antibody or functionalportion thereof which binds to the amino-terminal domain or portionthereof of mammalian CC-chemokine receptor 2, and particularly to anepitope comprising from about amino acid 1 to about amino acid 30 ofmammalian CC-chemokine receptor 2.

[0057] The invention also relates to a bispecific antibody, orfunctional fragment thereof (e.g., F(ab′)₂), which has the same orsimilar epitopic specificity as at least two of the antibodies describedherein (see, e.g., U.S. Pat. No. 5,141,736 (Iwasa et al.), U.S. Pat.Nos. 4,444,878, 5,292,668, 5,523,210 (all to Paulus et al.) and U.S.Pat. No. 5,496,549 (Yamazaki et al.). For example, a bispecific antibodyof the present invention can have the same or similar epitopicspecificity as mAb 1D9 and 8G2, e.g., binds the amino terminal domain,or portion thereof, of mammalian CCR2 protein.

[0058] Hybridoma cell lines producing antibodies according to thepresent invention were deposited on Jul. 17, 1998, on behalf ofLeukoSite, Inc., 215 First Street, Cambridge, Mass. 02142, U.S.A., atthe American Type Culture Collection, 10801 University Boulevard,Manassas, Va. 20110, U.S.A., under Accession Nos. HB-12549 (1D9) andHB-12550(8G2). The present invention also pertains to the hybridoma celllines deposited under ATCC Accession No. HB-12549 and ATCC Accession No.HB-12550, as well as to the monoclonal antibodies produced by thehybridoma cell lines deposited under ATCC Accession Nos. HB-12549 andHB-12550.

[0059] The antibodies of the present invention can be polyclonal ormonoclonal, and the term “antibody” is intended to encompass bothpolyclonal and monoclonal antibodies. Furthermore, it is understood thatmethods described herein which utilize 8G2 can also utilize functionalfragments (e.g., antigen-binding fragments) of 8G2, antibodies whichhave the same or similar epitopic specificity as 8G2, and combinationsthereof, optionally in combination with antibodies or fragments havingan epitopic specificity which is not the same as or similar to 8G2;similarly, methods described as utilizing 1D9 can also utilizefunctional fragments of 1D9, antibodies which have the same or similarepitopic specificity as 1D9, and combinations thereof, optionally incombination with antibodies or fragments having an epitopic specificitywhich is not the same as or similar to 1D9. Antibodies of the presentinvention can be raised against an appropriate immunogen, such asisolated and/or recombinant mammalian CCR2 protein or portion thereof,or synthetic molecules, such as synthetic peptides. In a preferredembodiment, cells which express receptor, such as transfected cells, canbe used as immunogens or in a screen for antibody which binds receptor.

[0060] The antibodies of the present invention, and fragments thereof,are useful in therapeutic, diagnostic and research applications asdescribed herein. The present invention encompasses an antibody orfunctional portion thereof of the present invention (e.g., mAb 1D9 or8G2, or antigen-binding fragments thereof) for use in therapy (includingprophylaxis) or diagnosis (e.g., of particular diseases or conditions asdescribed herein), and use of such antibodies or functional portionsthereof for the manufacture of a medicament for use in treatment ofdiseases or conditions as described herein.

[0061] Preparation of immunizing antigen, and polyclonal and monoclonalantibody production can be performed as described herein, or using othersuitable techniques. A variety of methods have been described (see e.g.,Kohler et al., Nature, 256: 495-497 (1975) and Eur. J. Immunol. 6:511-519 (1976); Milstein et al., Nature 266: 550-552 (1977); Koprowskiet al., U.S. Pat. No. 4,172,124; Harlow, E. and D. Lane, 1988,Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory: ColdSpring Harbor, N.Y.); Current Protocols In Molecular Biology, Vol. 2(Supplement 27, Summer '94), Ausubel, F. M. et al., Eds., (John Wiley &Sons: New York, N.Y.), Chapter 11, (1991)). Generally, a hybridoma canbe produced by fusing a suitable immortal cell line (e.g., a myelomacell line such as SP2/0) with antibody producing cells. The antibodyproducing cell, preferably those of the spleen or lymph nodes, areobtained from animals immunized with the antigen of interest. The fusedcells (hybridomas) can be isolated using selective culture conditions,and cloned by limiting dilution. Cells which produce antibodies with thedesired binding properties can be selected by a suitable assay (e.g.,ELISA).

[0062] Other suitable methods of producing or isolating antibodies whichbind CCR2, including human or artificial antibodies, can be used,including, for example, methods which select recombinant antibody (e.g.,single chain Fv or Fab) from a library, or which rely upon immunizationof transgenic animals (e.g., mice) capable of producing a repertoire ofhuman antibodies (see e.g., Jakobovits et al., Proc. Natl. Acad. Sci.USA, 90: 2551-2555 (1993); Jakobovits et al., Nature, 362: 255-258(1993); Lonberg et al., U.S. Pat. No. 5,545,806; Surani et al., U.S.Pat. No. 5,545,807).

[0063] Single chain antibodies, and chimeric, humanized or primatized(CDR-grafted) antibodies, as well as chimeric or CDR-grafted singlechain antibodies, and the like, comprising portions derived fromdifferent species, are also encompassed by the present invention and theterm “antibody”. The various portions of these antibodies can be joinedtogether chemically by conventional techniques, or can be prepared as acontiguous protein using genetic engineering techniques. For example,nucleic acids encoding a chimeric or humanized chain can be expressed toproduce a contiguous protein. See, e.g., Cabilly et al., U.S. Pat. No.4,816,567; Cabilly et al., European Patent No. 0,125,023 B1; Boss etal., U.S. Pat. No. 4,816,397; Boss et al., European Patent No. 0,120,694B1; Neuberger, M. S. et al., WO 86/01533; Neuberger, M. S. et al.,European Patent No. 0,194,276 B1; Winter, U.S. Pat. No. 5,225,539;Winter, European Patent No. 0,239,400 B1; and Queen et al., U.S. Pat.Nos. 5,585,089, 5,698,761 and 5,698,762. See also, Newman, R. et al.,BioTechnology, 10: 1455-1460 (1992), regarding primatized antibody, andLadner et al., U.S. Pat. No. 4,946,778 and Bird, R. E. et al., Science,242: 423-426 (1988)) regarding single chain antibodies.

[0064] In addition, functional fragments of antibodies, includingfragments of chimeric, humanized, primatized or single chain antibodies,can also be produced. Functional fragments of the foregoing antibodiesretain at least one binding function and/or modulation function of thefull-length antibody from which they are derived. Preferred functionalfragments retain an antigen-binding function of a correspondingfull-length antibody (e.g., the ability to bind a mammalian CCR2).Particularly preferred functional fragments retain the ability toinhibit one or more functions characteristic of a mammalian CCR2, suchas a binding activity, a signaling activity, and/or stimulation of acellular response. For example, in one embodiment, a functional fragmentcan inhibit the interaction of CCR2 with one or more of its ligands(e.g., MCP-1, MCP-2, MCP-3 and/or MCP-4) and/or can inhibit one or morereceptor-mediated functions, such as leukocyte trafficking, HIV entryinto cells, T cell activation, inflammatory mediator release and/orleukocyte degranulation.

[0065] For example, antibody fragments capable of binding to a mammalianCCR2 receptor or portion thereof, including, but not limited to, Fv,Fab, Fab′ and F(ab′)₂ fragments are encompassed by the invention. Suchfragments can be produced by enzymatic cleavage or by recombinanttechniques, for example. For instance, papain or pepsin cleavage cangenerate Fab or F(ab′)₂ fragments, respectively. Antibodies can also beproduced in a variety of truncated forms using antibody genes in whichone or more stop codons has been introduced upstream of the natural stopsite. For example, a chimeric gene encoding a F(ab′)₂ heavy chainportion can be designed to include DNA sequences encoding the CH₁ domainand hinge region of the heavy chain.

[0066] The present invention relates to a humanized immunoglobulin orantigen-binding fragment thereof having binding specificity for CCR2,comprising an antigen binding region of nonhuman origin (e.g., rodent)and at least a portion of an immunoglobulin of human origin (e.g., ahuman framework region, a human constant region or portion thereof). Inone embodiment, the humanized immunoglobulin includes an antigen bindingregion of nonhuman origin which binds CCR2 and a constant region derivedfrom a human constant region. In another embodiment, the humanizedimmunoglobulin which binds CCR2 comprises a complementarity determiningregion of nonhuman origin and a variable framework region of humanorigin, and optionally, a constant region of human origin. For example,the humanized immunoglobulin can comprise a heavy chain and a lightchain, wherein the light chain comprises a complementarity determiningregion derived from an antibody of nonhuman origin which binds CCR2 anda framework region derived from a light chain of human origin, and theheavy chain comprises a complementarity determining region derived froman antibody of nonhuman origin which binds CCR2 and a framework regionderived from a heavy chain of human origin.

[0067] In one embodiment, the humanized immunoglobulin can compete withmurine 1D9 or 8G2 monoclonal antibody for binding to human CCR2. In apreferred embodiment, the antigen-binding region of the humanizedimmunoglobulin (a) is derived from 1D9 monoclonal antibody (e.g., as ina humanized immunoglobulin comprising CDR1, CDR2 and CDR3 of the 1D9light chain and/or CDR1, CDR2 and CDR3 of the 1D9 heavy chain) or (b) isderived from 8G2 monoclonal antibody (e.g., as in a humanizedimmunoglobulin comprising CDR1, CDR2 and CDR3 of the 8G2 light chainand/or CDR1, CDR2 and CDR3 of the 8G2 heavy chain). Chimeric orCDR-grafted single chain antibodies are also encompassed by the termhumanized immunoglobulin.

[0068] The present invention also relates to a humanized immunoglobulinlight chain or antigen-binding fragment thereof or a humanizedimmunoglobulin heavy chain or antigen-binding fragment thereof. In oneembodiment, the invention relates to a humanized light chain comprisinga light chain CDR (i.e., one or more CDRs) of nonhuman origin and ahuman light chain framework region. In another embodiment, the presentinvention relates to a humanized immunoglobulin heavy chain comprising aheavy chain CDR (i.e., one or more CDRs) of nonhuman origin and a humanheavy chain framework region. The CDRs can be derived from a nonhumanimmunoglobulin.

[0069] Naturally occurring immunoglobulins have a common core structurein which two identical light chains (about 24 kD) and two identicalheavy chains (about 55 or 70 kD) form a tetramer. The amino-terminalportion of each chain is known as the variable (V) region and can bedistinguished from the more conserved constant (C) regions of theremainder of each chain. Within the variable region of the light chainis a C-terminal portion known as the J region. Within the variableregion of the heavy chain, there is a D region in addition to the Jregion. Most of the amino acid sequence variation in immunoglobulins isconfined to three separate locations in the V regions known ashypervariable regions or complementarity determining regions (CDRs)which are directly involved in antigen binding. Proceeding from theamino-terminus, these regions are designated CDR1, CDR2 and CDR3,respectively. The CDRs are held in place by more conserved frameworkregions (FRs). Proceeding from the amino-terminus, these regions aredesignated FR1, FR2, FR3, and FR4, respectively. The locations of CDRand FR regions and a numbering system have been defined by Kabat et al.(Kabat et al., Sequences of Proteins of Immunological Interest, FifthEdition, U.S. Department of Health and Human Services, U.S. GovernmentPrinting Office (1991)).

[0070] Human immunoglobulins can be divided into classes and subclasses,depending on the isotype of the heavy chain. The classes include IgG,IgM, IgA, IgD and IgE, in which the heavy chains are of the gamma (γ),mu (μ), alpha (α), delta (δ) or epsilon (ε) type, respectively.Subclasses include IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2, in which theheavy chains are of the γ1, γ2, γ3, γ4, α1 and α2 type, respectively.Human immunoglobulin molecules of a selected class or subclass maycontain either a kappa (κ) or lambda (λ) light chain. See e.g., Cellularand Molecular Immunology, Wonsiewicz, M. J., Ed., Chapter 45, pp. 41-50,W. B. Saunders Co, Philadelphia, Pa. (1991); Nisonoff, A., Introductionto Molecular Immunology, 2nd Ed., Chapter 4, pp. 45-65, SinauerAssociates, Inc., Sunderland, Mass. (1984).

[0071] The term “immunoglobulin” as used herein includes wholeantibodies and biologically functional fragments thereof. Suchbiologically functional fragments retain at least one antigen-bindingfunction of a corresponding full-length antibody (e.g., specificity forCCR2 of antibody 1D9), and preferably, retain the ability to inhibit theinteraction of CCR2 with one or more of its ligands (e.g., HIV, MCP-1,MCP-2, MCP-3, MCP-4). Examples of biologically functional antibodyfragments which can be used include fragments capable of binding toCCR2, such as single chain antibodies, Fv, Fab, Fab′ and F(ab′)₂fragments. Such fragments can be produced by enzymatic cleavage or byrecombinant techniques. For instance, papain or pepsin cleavage can beused to generate Fab or F(ab′)₂ fragments, respectively. Antibodies canalso be produced in a variety of truncated forms using antibody genes inwhich one or more stop codons have been introduced upstream of thenatural stop site. For example, a chimeric gene encoding the heavy chainof an F(ab′)₂ fragment can be designed to include DNA sequences encodingthe CH₁ domain and hinge region of the heavy chain. As used herein, anantigen-binding fragment of a humanized immunoglobulin heavy or lightchain is intended to mean a fragment which binds to an antigen whenpaired with a complementary chain. That is, an antigen-binding fragmentof a humanized light chain will bind to an antigen when paired with aheavy chain (e.g., murine, chimeric, humanized) comprising a variableregion, and an antigen-binding fragment of a humanized heavy chain willbind to an antigen when paired with a light chain (e.g., murine,chimeric, humanized) comprising a variable region.

[0072] The term “humanized immunoglobulin” as used herein refers to animmunoglobulin comprising portions of immunoglobulins of differentorigin, wherein at least one portion is of human origin. For example,the humanized antibody can comprise portions derived from animmunoglobulin of nonhuman origin with the requisite specificity, suchas a mouse, and from immunoglobulin sequences of human origin (e.g.,chimeric immunoglobulin), joined together chemically by conventionaltechniques (e.g., synthetic) or prepared as a contiguous polypeptideusing genetic engineering techniques (e.g., DNA encoding the proteinportions of the chimeric antibody can be expressed to produce acontiguous polypeptide chain). Another example of a humanizedimmunoglobulin of the present invention is an immunoglobulin containingone or more immunoglobulin chains comprising a CDR derived from anantibody of nonhuman origin and a framework region derived from a lightand/or heavy chain of human origin (e.g., CDR-grafted antibodies with orwithout framework changes). Chimeric or CDR-grafted single chainantibodies are also encompassed by the term humanized immunoglobulin.See, e.g., Cabilly et al., U.S. Pat. No. 4,816,567; Cabilly et al.,European Patent No. 0,125,023 B1; Boss et al., U.S. Pat. No. 4,816,397;Boss et al., European Patent No. 0,120,694 B1; Neuberger, M. S. et al.,WO 86/01533; Neuberger, M. S. et al., European Patent No. 0,194,276 B1;Winter, U.S. Pat. No. 5,225,539; Winter, European Patent No. 0,239,400B1; Padlan, E. A. et al., European Patent Application No. 0,519,596 A1.See also, Ladner et al., U.S. Pat. No. 4,946,778; Huston, U.S. Pat. No.5,476,786; and Bird, R. E. et al., Science, 242: 423-426 (1988)),regarding single chain antibodies.

[0073] For example, humanized immunoglobulins can be produced usingsynthetic and/or recombinant nucleic acids to prepare genes (e.g., cDNA)encoding the desired humanized chain. For example, nucleic acid (e.g.,DNA) sequences coding for humanized variable regions can be constructedusing PCR mutagenesis methods to alter DNA sequences encoding a human orhumanized chain, such as a DNA template from a previously humanizedvariable region (see e.g., Kamman, M., et al., Nucl. Acids Res., 1 7:5404 (1989)); Sato, K., et al., Cancer Research, 53: 851-856 (1993);Daugherty, B. L. et al., Nucleic Acids Res., 19(9): 2471-2476 (1991);and Lewis, A. P. and J. S. Crowe, Gene, 101: 297-302 (1991)). Usingthese or other suitable methods, variants can also be readily produced.In one embodiment, cloned variable regions can be mutagenized, andsequences encoding variants with the desired specificity can be selected(e.g., from a phage library; see e.g., Krebber et al., U.S. Pat. No.5,514,548; Hoogenboom et al., WO 93/06213, published Apr. 1, 1993)).

[0074] The antigen binding region of the humanized immunoglobulin (thenonhuman portion) can be derived from an immunoglobulin of nonhumanorigin (referred to as a donor immunoglobulin) having bindingspecificity for CCR2. For example, a suitable antigen binding region canbe derived from the murine monoclonal antibody 1D9. Other sourcesinclude CCR2-specific antibodies obtained from nonhuman sources, such asrodent (e.g., mouse, rat), rabbit, pig goat or non-human primate (e.g.,monkey). Additionally, other polyclonal or monoclonal antibodies, suchas antibodies which bind to the same or similar epitope as the 1D9antibody, can be made (e.g., Kohler et al., Nature, 256:495-497 (1975);Harlow et al., 1988, Antibodies: A Laboratory Manual, (Cold SpringHarbor, N.Y.); and Current Protocols in Molecular Biology, Vol. 2(Supplement 27, Summer '94), Ausubel et al., Eds. (John Wiley & Sons:New York, N.Y.), Chapter 11 (1991)).

[0075] For example, antibodies can be raised against an appropriateimmunogen in a suitable mammal (e.g., a mouse, rat, rabbit or sheep).Cells bearing CCR2, membrane fractions containing CCR2, and immunogenicfragments of CCR2 are examples of suitable immunogens.Antibody-producing cells (e.g., a lymphocyte) can be isolated from, forexample, the lymph nodes or spleen of an immunized animal. The cells canthen be fused to a suitable immortalized cell (e.g., a myeloma cellline), thereby forming a hybridoma. Fused cells can be isolatedemploying selective culturing techniques. Cells which produce antibodieswith the desired specificity can be selected by a suitable assay (e.g.,ELISA). Immunoglobulins of nonhuman origin having binding specificityfor CCR2 can also be obtained from antibody libraries (e.g., a phagelibrary comprising nonhuman Fab molecules).

[0076] In one embodiment, the antigen binding region of the humanizedimmunoglobulin comprises a CDR of nonhuman origin. In this embodiment,the humanized immunoglobulin having binding specificity for CCR2comprises at least one CDR of nonhuman origin. For example, CDRs can bederived from the light and heavy chain variable regions ofimmunoglobulins of nonhuman origin, such that a humanized immunoglobulinincludes substantially heavy chain CDR1, CDR2 and/or CDR3, and/or lightchain CDR1, CDR2 and/or CDR3, from one or more immunoglobulins ofnonhuman origin, and the resulting humanized immunoglobulin has bindingspecificity for CCR2. Preferably, all three CDRs of a selected chain aresubstantially the same as the CDRs of the corresponding chain of adonor, and more preferably, all three CDRs of the light and heavy chainsare substantially the same as the CDRs of the corresponding donor chain.In one embodiment, the invention relates to an immunoglobulin havingbinding specificity for CCR2 comprising a humanized light chain orantigen-binding fragment thereof comprising CDR 1, CDR2 and CDR3 of thelight chain of the 1D9 antibody and a heavy chain, e.g., a human heavychain. The invention also includes an immunoglobulin having bindingspecificity for CCR2 comprising a humanized heavy chain orantigen-binding fragment thereof comprising CDR1, CDR2 and CDR3 of theheavy chain of the 1D9 antibody and a light chain, e.g., a human lightchain.

[0077] The invention also relates to an immunoglobulin having bindingspecificity for CCR2 comprising a light chain and a heavy chain, whereinthe light chain comprises at least 1 CDR of an antibody of non-humanorigin (e.g., 1D9) and framework and constant regions of human origin(e.g., SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14 and SEQ ID NO: 15),and wherein the heavy chain comprises a variable region of non-humanorigin (e.g., from 1D9) and a constant region of human origin. Theinvention also provides antigen-binding fragments of theseimmunoglobulins. The invention also relates to an immunoglobulin havingbinding specificity for CCR2 comprising a light chain and a heavy chain,wherein the light chain comprises a variable chain of non-human origin(e.g., from 1D9) and a constant region of human origin, and wherein theheavy chain comprises at least 1 CDR of an antibody of non-human origin(e.g., 1D9) and framework and constant regions of human origin (e.g.,SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO:19 and SEQ ID NO: 20). Theinvention also provides antigen-binding fragments of theseimmunoglobulins.

[0078] The portion of the humanized immunoglobulin or immunoglobulinchain which is of human origin (the human portion) can be derived fromany suitable human immunoglobulin or immunoglobulin chain. For example,a human constant region or portion thereof, if present, can be derivedfrom the κ or λ light chains, and/or the γ (e.g., γ1, γ2, γ3, γ4), μ, α(e.g., α1, α2), δ or ε heavy chains of human antibodies, includingallelic variants. A particular constant region (e.g., IgG1), variant orportions thereof can be selected in order to tailor effector function.For example, a mutated constant region (variant) can be incorporatedinto a fusion protein to minimize binding to Fc receptors and/or abilityto fix complement (see e.g., Winter et al., GB 2,209,757 B; Morrison etal., WO 89/07142; Morgan et al., WO 94/29351, Dec. 22, 1994).

[0079] If present, human framework regions (e.g., of the light chainvariable region) are preferably derived from a human antibody variableregion having sequence similarity to the analogous or equivalent region(e.g., light chain variable region) of the antigen binding region donor.Other sources of framework regions for portions of human origin of ahumanized immunoglobulin include human variable consensus sequences(see, e.g., Kettleborough, C. A. et al., Protein Engineering 4:773-783(1991); Carter et al., WO 94/04679, published Mar. 3, 1994)). Forexample, the sequence of the antibody or variable region used to obtainthe nonhuman portion can be compared to human sequences as described inKabat et al., Sequences of Proteins of Immunological Interest, FifthEdition, U.S. Department of Health and Human Services, U.S. GovernmentPrinting Office (1991). In a particularly preferred embodiment, theframework regions of a humanized immunoglobulin chain are derived from ahuman variable region having at least about 60% overall sequenceidentity, preferably at least about 70% overall sequence identity andmore preferably at least about 85% overall sequence identity, with thevariable region of the nonhuman donor (e.g., murine antibody 1D9). Ahuman portion can also be derived from a human antibody having at leastabout 65% sequence identity, and preferably at least about 70% sequenceidentity, within the particular portion (e.g., FR) being used, whencompared to the equivalent portion (e.g., FR) of the nonhuman donor.

[0080] In one embodiment, the humanized immunoglobulin comprises atleast one of the framework regions (FR) derived from one or more chainsof an antibody of human origin. Thus, the FR can include a FR1 and/orFR2 and/or FR3 and/or FR4 derived from one or more antibodies of humanorigin. Preferably, the human portion of a selected humanized chainincludes FR1, FR2, FR3 and FR4 derived from a variable region of humanorigin (e.g., from a human immunoglobulin chain, from a human consensussequence).

[0081] The immunoglobulin portions of nonhuman and human origin for usein the present invention have sequences identical to immunoglobulins orimmunoglobulin portions from which they are derived or to variantsthereof. Such variants include mutants differing by the addition,deletion, or substitution of one or more residues. As indicated above,the CDRs which are of nonhuman origin are substantially the same as inthe nonhuman donor, and preferably are identical to the CDRs of thenonhuman donor. As described in Example 2, changes in the frameworkregion, such as those which substitute a residue of the framework regionof human origin with a residue from the corresponding position of thedonor, can be made. One or more mutations in the framework region can bemade, including deletions, insertions and substitutions of one or moreamino acids. Several such substitutions are described in the design ofhumanized 1D9 antibodies in Example 2. For a selected humanized antibodyor chain, framework mutations can be designed as described herein.Preferably, the humanized immunoglobulins can bind CCR2 with an affinitysimilar to or better than that of the nonhuman donor. Variants can beproduced by a variety of suitable methods, including mutagenesis ofnonhuman donor or acceptor human chains.

[0082] The humanized immunoglobulins of the present invention havebinding specificity for human CCR2. In a preferred embodiment, thehumanized immunoglobulin of the present invention has at least onefunctional characteristic of murine antibody 1D9, such as bindingfunction (e.g., having specificity for CCR2, having the same or similarepitopic specificity), and/or inhibitory function (e.g., the ability toinhibit CCR2-dependent function in vitro and/or in vivo, such as theability to inhibit the binding of a cell bearing CCR2 to a ligandthereof (e.g., a chemokine)). Thus, preferred humanized immunoglobulinscan have the binding specificity of the murine antibody 1D9, theepitopic specificity of murine antibody 1D9 (e.g., can compete withmurine 1D9, a chimeric 1D9 antibody, or humanized 1D9 for binding toCCR2 (e.g., on a cell bearing CCR2)), and/or inhibitory function ofmurine antibody 1D9.

[0083] The binding function of a humanized immunoglobulin having bindingspecificity for CCR2 can be detected by standard immunological methods,for example using assays which monitor formation of a complex betweenhumanized immunoglobulin and CCR2 (e.g., a membrane fraction comprisingCCR2, on a cell bearing CCR2, human cell line or recombinant host cellcomprising nucleic acid encoding CCR2 which expresses CCR2). Bindingand/or adhesion assays or other suitable methods can also be used inprocedures for the identification and/or isolation of humanizedimmunoglobulins (e.g., from a library) with the requisite specificity(e.g., an assay which monitors adhesion between a cell bearing CCR2 anda ligand thereof (e.g., HIV, MCP-1, MCP-2, MCP-3, MCP-4), or othersuitable methods.

[0084] The immunoglobulin portions of nonhuman and human origin for usein the present invention include light chains, heavy chains and portionsof light and heavy chains. These immunoglobulin portions can be obtainedor derived from immunoglobulins (e.g., by de novo synthesis of aportion), or nucleic acid molecules encoding an immunoglobulin or chainthereof having the desired property (e.g., binding CCR2, sequencesimilarity) can be produced and expressed. Humanized immunoglobulinscomprising the desired portions (e.g., antigen binding region, CDR, FR,C region) of human and nonhuman origin can be produced using syntheticand/or recombinant nucleic acids to prepare genes (e.g., cDNA) encodingthe desired humanized chain. To prepare a portion of a chain, one ormore stop codons can be introduced at the desired position. For example,nucleic acid (e.g., DNA) sequences coding for newly designed humanizedvariable regions can be constructed using PCR mutagenesis methods toalter existing DNA sequences (see e.g., Kamman, M., et al., Nucl. AcidsRes. 17:5404 (1989)). PCR primers coding for the new CDRs can behybridized to a DNA template of a previously humanized variable regionwhich is based on the same, or a very similar, human variable region(Sato, K., et al., Cancer Research 53:851-856 (1993)). If a similar DNAsequence is not available for use as a template, a nucleic acidcomprising a sequence encoding a variable region sequence can beconstructed from synthetic oligonucleotides (see e.g., Kolbinger, F.,Protein Engineering 8:971-980 (1993)). A sequence encoding a signalpeptide can also be incorporated into the nucleic acid (e.g., onsynthesis, upon insertion into a vector). If the natural signal peptidesequence is unavailable, a signal peptide sequence from another antibodycan be used (see, e.g., Kettleborough, C. A., Protein Engineering4:773-783 (1991)). Using these methods, methods described herein orother suitable methods, variants can be readily produced. In oneembodiment, cloned variable regions can be mutagenized, and sequencesencoding variants with the desired specificity can be selected (e.g.,from a phage library; see e.g., Krebber et al., U.S. Pat. No. 5,514,548;Hoogenboom et al., WO 93/06213, published Apr. 1, 1993)).

[0085] The invention relates to a humanized immunoglobulin light chainor antigen-binding fragment thereof, said light chain or antigen-bindingfragment thereof having an amino acid sequence comprising at least afunctional portion of the light chain variable region amino acidsequence of SEQ ID NO: 9. In a preferred embodiment, the amino acidsequence comprises at least one, preferably two, and more preferablythree of the CDRs of SEQ ID NO: 9. The invention also relates to ahumanized immunoglobulin heavy chain or antigen-binding fragmentthereof, said heavy chain or antigen-binding fragment thereof having anamino acid sequence comprising at least a functional portion of theheavy chain variable region amino acid sequence shown in SEQ ID NO: 10.In a preferred embodiment, the amino acid sequence comprises at leastone, preferably two, and more preferably three of the CDRs of SEQ ID NO:10. It is noted that all murine sequences described herein are derivedfrom Mus musculus.

[0086] According to one embodiment of the invention, a humanizedimmunoglobulin light chain or antigen-binding fragment thereof havingbinding specificity for CCR2 can comprise an amino acid sequenceselected from the group consisting of SEQ ID NO: 12, SEQ ID NO: 13, SEQID NO: 14, and SEQ ID NO: 15. According to another embodiment of theinvention, a humanized immunoglobulin heavy chain or antigen-bindingfragment thereof having binding specificity for CCR2 can comprise anamino acid sequence selected from the group consisting of SEQ ID NO: 17,SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20. In a particularembodiment, a humanized immunoglobulin of the invention can compriseboth a light chain or antigen-binding fragment thereof having bindingspecificity for CCR2, comprising an amino acid sequence selected fromthe group consisting of SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, andSEQ ID NO: 15, and a heavy chain or antigen-binding fragment thereofhaving binding specificity for CCR2 comprising an amino acid sequenceselected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 18, SEQID NO: 19, and SEQ ID NO: 20. In one embodiment, the humanizedimmunoglobulin light chain or antigen-binding fragment thereof havingbinding specificity for CCR2 can be encoded by a nucleic acid moleculecomprising SEQ ID NO: 98. In another embodiment, the humanizedimmunoglobulin heavy chain or antigen-binding fragment thereof havingbinding specificity for CCR2 can be encoded by a nucleic acid moleculecomprising SEQ ID NO: 97.

[0087] The invention also relates to a chimeric immunoglobulin orantigen-binding fragment thereof having binding specificity for CCR2comprising a light chain variable region of nonhuman origin and a humanconstant region (e.g., a light chain constant region). The inventionfurther relates to a chimeric immunoglobulin or antigen-binding fragmentthereof having binding specificity for CCR2 comprising a heavy chainvariable region of nonhuman origin and a human constant region (e.g., aheavy chain constant region). In another embodiment, the chimericimmunoglobulin or antigen-binding fragment thereof having bindingspecificity for CCR2 comprises a light chain variable chain region ofnonhuman origin and a heavy chain variable region of nonhuman origin andfurther comprises a human constant region (e.g., a human light chainconstant region and/or a human heavy chain constant region).

[0088] Nucleic Acids and Constructs

[0089] The present invention also relates to isolated and/or recombinant(including, e.g., essentially pure) nucleic acid molecules comprisingnucleic acid sequences which encode a humanized immunoglobulin orhumanized immunoglobulin light or heavy chain of the present invention.

[0090] Nucleic acid molecules referred to herein as “isolated” arenucleic acid molecules which have been separated away from the nucleicacids of the genomic DNA or cellular RNA of their source of origin(e.g., as it exists in cells or in a mixture of nucleic acids such as alibrary), and include nucleic acid molecules obtained by methodsdescribed herein or other suitable methods, including essentially purenucleic acid molecules, nucleic acid molecules produced by chemicalsynthesis, by combinations of biological and chemical methods, andrecombinant nucleic acid molecules which are isolated (see e.g.,Daugherty, B. L. et al., Nucleic Acids Res., 19(9): 2471-2476 (1991);Lewis, A. P. and J. S. Crowe, Gene, 101: 297-302 (1991)).

[0091] Nucleic acid molecules referred to herein as “recombinant” arenucleic acid molecules which have been produced by recombinant DNAmethodology, including those nucleic acid molecules that are generatedby procedures which rely upon a method of artificial recombination, suchas the polymerase chain reaction (PCR) and/or cloning into a vectorusing restriction enzymes. “Recombinant” nucleic acid molecules are alsothose that result from recombination events that occur through thenatural mechanisms of cells, but are selected for after the introductionto the cells of nucleic acids designed to allow and make probable adesired recombination event.

[0092] The present invention also relates more specifically to isolatedand/or recombinant nucleic acid molecules comprising a nucleotidesequence which encodes a humanized 1D9 immunoglobulin (i.e., a humanizedimmunoglobulin of the present invention in which the nonhuman portion isderived from the murine monoclonal antibody 1D9) or chain thereof. Inone embodiment, the light chain comprises three complementaritydetermining regions derived from the light chain of the 1D9 antibody,and the heavy chain comprises three complementarity determining regionsderived from the heavy chain of the 1D9 antibody. Such nucleic acidmolecules include, for example, (a) a nucleic acid molecule comprising asequence which encodes a polypeptide comprising the amino acid sequenceof the heavy chain variable region of a humanized 1D9 immunoglobulin(e.g., heavy chain variable region of FIGS. 8 and 21) (e.g., nucleotides58-411 of SEQ ID NO: 96); (b) a nucleic acid molecule comprising asequence which encodes a polypeptide comprising the amino acid sequenceof the light chain variable region of a humanized 1D9 immunoglobulin(e.g., light chain variable region of FIGS. 7 and 22) (e.g., nucleotides52-390 of SEQ ID NO: 95); (c) a nucleic acid molecule comprising asequence which encodes at least a functional portion of the light orheavy chain variable region of a humanized 1D9 immunoglobulin (e.g., aportion sufficient for antigen binding of a humanized immunoglobulinwhich comprises said chain). Due to the degeneracy of the genetic code,a variety of nucleic acids can be made which encode a selectedpolypeptide. In one embodiment, the nucleic acid comprises thenucleotide sequence of the variable region as set forth or substantiallyas set forth in FIG. 21 or as set forth or substantially as set forth inFIG. 22, including double or single-stranded polynucleotides. (Althoughvarious figures may illustrate polypeptides which are larger than thevariable region (i.e., include a signal peptide coding sequence or aportion of a constant region coding sequence), reference to the variableregion of a particular figure is meant to include the variable regionportion of the sequence shown). Isolated and/or recombinant nucleic acidmolecules meeting these criteria can comprise nucleic acid moleculesencoding sequences identical to sequences of humanized 1D9 antibody orvariants thereof as discussed above.

[0093] Nucleic acid molecules of the present invention can be used inthe production of humanized immunoglobulins having binding specificityfor CCR2. For example, a nucleic acid molecule (e.g., DNA) encoding ahumanized immunoglobulin of the present invention can be incorporatedinto a suitable construct (e.g., a vector) for further manipulation ofsequences or for production of the encoded polypeptide in suitable hostcells.

[0094] Targeting Molecules

[0095] The invention also relates to targeting molecules which caneffectuate the interaction of a CCR2-expressing cell with a target cell.The targeting molecule includes a first binding moiety which can bindmammalian CCR2, and a second binding moiety which can bind a moleculeexpressed on the surface of a target cell. Preferred target cellsinclude tumor cells and virus infected cells. A variety of moleculeswhich are expressed at higher levels or uniquely on tumor cells (e.g.,tumor antigens, such as Lewis Y, HER-2/neu, disialoganglioside G3,carcinoembrionic antigen, CD30) and/or virus infected cells (e.g., viralantigens, such as influenza virus hemagglutinin, Epstein-Barr virusLMP-1, hepatitis C virus E2 glycoprotein, HIV gp160, HIV gp 120) areknown in the art. The targeting molecule can contain any suitablebinding second moiety which binds to a molecule expressed on a desiredtarget cell (see, for example Ring, U.S. Pat. No. 5,948,647, the entireteachings of which are incorporated herein by reference). Suitablebinding moieties include, for example, proteins and peptides (includingpost-translationally modified forms e.g., glycosylated, phosphorylated,lipidated), sugars, lipids, peptidomimetics, small organic molecules,nucleic acids and other agents which bind mammalian CCR2 or a moleculeexpressed on the surface of a target cell. Suitable binding moieties canbe identified using any suitable method, such as the binding assaysdescribed herein.

[0096] In a preferred embodiment, the first binding moiety can be, forexample, a humanized immunoglobulin of the invention which bindsmammalian CCR2 or antigen-binding fragment thereof (e.g., Fab, Fv, Fab′,F(ab)′₂). The second binding moiety can be, for example, an antibody(e.g., a second humanized immunoglobulin) or antigen-binding fragmentthereof which binds to a molecule expressed on the target cell orantigen binding fragment thereof. Where the targeting molecule comprisesa first binding moiety which is a humanized anti-CCR2 immunoglobulin orantigen-binding fragment thereof, it is preferred that the humanizedanti-CCR2 immunoglobulin does not inhibit binding of ligand to CCR2.

[0097] The first binding moiety can be directly or indirectly bonded tothe second binding moiety through a variety of suitable linkages. Forexample, when the first binding moiety and the second binding moiety areboth proteins or peptides, the moieties can be part of a contiguouspolypeptide (i.e., a fusion protein). Where the targeting molecule is afusion protein, the first and second binding moieties can be arranged onthe polypeptide in any suitable configuration. The first and secondbinding moieties can be indirectly bonded through a (i.e., one or more)peptide linker, or bonded directly to each other through a peptide bond.

[0098] Where the binding moieties are not part of a contiguouspolypeptide they can be directly bonded by a chemical bond formed byreaction of a functional group (or activated derivative thereof) on thefirst moiety with a second functional group (or activated derivativethereof) on the second moiety. For example, two thiols can react to forma disulfide bond and an amine can react with a carboxylic acid or acylhalide to form an amide. A variety of other suitable reactions which canbe used are known in the art (see, for example, Hermanson, G. T.,Bioconjugate Techniques, Academic Press: San Diego, Calif. (1996)). Thebinding moieties can be indirectly bonded through a suitable linker(e.g., a peptide linker). Generally, a linker contains two reactivegroups which can react to form bonds with the first binding moietyand/or the second binding moiety. Linkers which contain two differentreactive groups (e.g., a heterobifunctional linker) can be used toselectively conjugate the first binding moiety to the second bindingmoiety. Many linkers which are suitable for forming conjugates betweenproteins, nucleic acids, peptides, vitamins, sugars, lipids, smallorganic molecules and other suitable agents are known (see, for example,U.S. Pat. Nos. 5,856,571, 5,880,270; Hermanson, G. T., BioconjugateTechniques, Academic Press: San Diego, Calif. (1996)).

[0099] Preferably, the independent activities of the binding moieties(e.g., binding activities, chemoattractant activity) of the targetingmolecule are not significantly different from the activities of thebinding moieties as separate molecular entities. For example, where thefirst binding moiety is a humanized immunoglobulin or antigen-bindingfragment that binds CCR2, the targeting molecule can bind to CCR2 withan affinity which is within a factor of about 1000, preferably within afactor of 100, more preferably within a factor of 10 or substantiallythe same as the affinity of the free antibody or antigen-bindingfragment. Target molecules with these preferred characteristics can beprepared using any suitable method. The resulting targeting molecule canthen be assayed for binding (e.g., by ELISA) and for chemoattractantactivity.

[0100] In one embodiment, the targeting molecule is a bispecifichumanized antibody or bispecific antigen-binding fragment thereof (e.g.,F(ab′)₂) which has specificity for mammalian CCR2 and a moleculeexpressed on a target cell (e.g., tumor antigen, viral antigen).Bispecific antibodies can be secreted by triomas and hybrid hybridomas.The supernatants of triomas and hybrid hybridomas can be assayed forbispecific antibody using a suitable assay (e.g., ELISA), and bispecificantibodies can be purified using conventional methods. These antibodiescan then be humanized according to methods described herein. Thus, theinvention provides a targeting molecule which is a humanized bispecificantibody having binding specificity for CCR2 and an antigen expressed ona target cell, or a bivalent antigen-binding fragment of the bispecificantibody. The invention also relates to a method of effectuating theinteraction of a CCR2-bearing cell with a target cell in a patient,comprising administering to the patient an effective amount of atargeting molecule which is a humanized bispecific antibody havingbinding specificity for CCR2 and an antigen expressed on a target cell,or a bivalent antigen-binding fragment of the bispecific antibody.

[0101] Method of Producing Humanized Immunoglobulins Having Specificityfor CCR2

[0102] Another aspect of the invention relates to a method of preparinga humanized immunoglobulin which has binding specificity for CCR2. Thehumanized immunoglobulin can be obtained, for example, by the expressionof one or more recombinant nucleic acids encoding a humanizedimmunoglobulin having binding specificity for CCR2 in a suitable hostcell, for example.

[0103] Constructs or expression vectors suitable for the expression of ahumanized immunoglobulin having binding specificity for CCR2 are alsoprovided. The constructs can be introduced into a suitable host cell,and cells which express a humanized immunoglobulin of the presentinvention can be produced and maintained in culture. Suitable host cellscan be prokaryotic, including bacterial cells such as E. coli, B.subtilis and or other suitable bacteria, or eucaryotic, such as fungalor yeast cells (e.g., Pichia pastoris, Aspergillus species,Saccharomyces cerevisiae, Schizosaccharomyces pombe, Neurospora crassa),or other lower eucaryotic cells, and cells of higher eucaryotes such asthose from insects (e.g., Sf9 insect cells (WO 94/26087, O'Connor,published Nov. 24, 1994)) or mammals (e.g., COS cells, such as COS-1(ATCC Accession No. CRL-1650) and COS-7 (ATCC Accession No. CRL-1651),CHO (e.g., ATCC Accession No. CRL-9096), 293 (ATCC Accession No.CRL-1573), HeLa (ATCC Accession No. CCL-2), CV1 (ATCC Accession No.CCL-70), WOP (Dailey et al., J. Virol. 54:739-749 (1985)), 3T3, 293T(Pear et al., Proc. Natl. Acad. Sci. U.S.A., 90:8392-8396 (1993)), NSOcells, SP2/0, HuT 78 cells, and the like (see, e.g., Ausubel, F. M. etal., eds. Current Protocols in Molecular Biology, Greene PublishingAssociates and John Wiley & Sons Inc., (1993)).

[0104] Host cells which produce a humanized immunoglobulin havingbinding specificity for CCR2 can be produced as follows. For example, anucleic acid encoding all or part of the coding sequence for the desiredhumanized immunoglobulin can be inserted into a nucleic acid vector,e.g., a DNA vector, such as a plasmid, virus or other suitable repliconfor expression. A variety of vectors are available, including vectorswhich are maintained in single copy or multiple copy, or which becomeintegrated into the host cell chromosome.

[0105] Suitable expression vectors can contain a number of components,including, but not limited to one or more of the following: an origin ofreplication; a selectable marker gene; one or more expression controlelements, such as a transcriptional control element (e.g., a promoter,an enhancer, terminator), and/or one or more translation signals; asignal sequence or leader sequence for membrane targeting or secretion.In a construct, a signal sequence can be provided by the vector or othersource. For example, the transcriptional and/or translational signals ofan immunoglobulin can be used to direct expression.

[0106] A promoter can be provided for expression in a suitable hostcell. Promoters can be constitutive or inducible. For example, apromoter can be operably linked to a nucleic acid encoding a humanizedimmunoglobulin or immunoglobulin chain, such that it directs expressionof the encoded polypeptide. A variety of suitable promoters forprocaryotic (e.g., lac, tac, T3, T7 promoters for E. coli) andeucaryotic (e.g., yeast alcohol dehydrogenase (ADH1), SV40, CMV) hostsare available.

[0107] In addition, the expression vectors typically comprise aselectable marker for selection of host cells carrying the vector, and,in the case of replicable expression vector, an origin or replication.Genes encoding products which confer antibiotic or drug resistance arecommon selectable markers and may be used in procaryotic (e.g.,β-lactamase gene (ampicillin resistance), Tet gene for tetracyclineresistance) and eucaryotic cells (e.g., neomycin (G418 or geneticin),gpt (mycophenolic acid), ampicillin, or hygromycin resistance genes).Dihydrofolate reductase marker genes permit selection with methotrexatein a variety of hosts. Genes encoding the gene product of auxotrophicmarkers of the host (e.g., LEU2, URA3, HIS3) are often used asselectable markers in yeast. Use of viral (e.g., baculovirus) or phagevectors, and vectors which are capable of integrating into the genome ofthe host cell, such as retroviral vectors, are also contemplated. In oneembodiment, the vector is pLKTOK38. The present invention also relatesto cells carrying these expression vectors. An expression vectorcomprising a fused gene encoding a humanized immunoglobulin light chain,said gene comprising a nucleotide sequence encoding a CDR derived from alight chain of a nonhuman antibody having binding specificity for CCR2and a framework region derived from a light chain of human origin.

[0108] Thus, the invention includes an expression vector comprising agene encoding a humanized immunoglobulin light chain, said genecomprising a nucleotide sequence encoding a CDR derived from a lightchain of a nonhuman antibody having binding specificity for CCR2 and aframework region derived from a light chain of human origin. Theinvention also relates to an expression vector comprising a geneencoding a humanized immunoglobulin heavy chain, said gene comprising anucleotide sequence encoding a CDR derived from a heavy chain of anonhuman antibody having binding specificity for CCR2 and a frameworkregion derived from a heavy chain of human origin. In on embodiment, thenonhuman antibody is murine antibody 1D9. The invention also includeshost cells comprising the expression vectors of the invention. Theinvention also relates to an isolated or recombinant gene encoding ahumanized immunoglobulin light or heavy chain comprising a first nucleicacid sequence encoding an antigen binding region derived from murinemonoclonal antibody 1D9; and a second nucleic acid sequence encoding atleast a portion of a constant region of an immunoglobulin of humanorigin.

[0109] The invention also relates to a host cell (e.g., which expressesa humanized immunoglobulin or an antigen binding fragment thereof havingspecificity for CCR2) comprising a first recombinant nucleic acidmolecule encoding a humanized immunoglobulin light chain or fragmentthereof and a second recombinant nucleic acid molecule encoding ahumanized immunoglobulin heavy chain or fragment thereof, wherein saidfirst nucleic acid molecule comprises a nucleotide sequence encoding aCDR derived from the light chain of murine antibody 1D9 and a frameworkregion derived from a light chain of human origin, and wherein saidsecond nucleic acid molecule comprises a nucleotide sequence encoding aCDR derived from the heavy chain of murine antibody 1D9 and a frameworkregion derived from a heavy chain of human origin. The invention alsoincludes a method of preparing a humanized immunoglobulin orantigen-binding fragment thereof comprising maintaining a host cell ofthe invention under conditions appropriate for expression of a humanizedimmunoglobulin, whereby humanized immunoglobulin chains are expressedand a humanized immunoglobulin or antigen-binding fragment thereofhaving specificity for CCR2 is produced. The method can further comprisethe step of isolating the humanized immunoglobulin or fragment thereof.

[0110] For example, a nucleic acid molecule (i.e., one or more nucleicacid molecules) encoding the heavy and light chains of a humanizedimmunoglobulin having binding specificity for CCR2, or a construct(i.e., one or more constructs) comprising such nucleic acid molecule(s),can be introduced into a suitable host cell by a method appropriate tothe host cell selected (e.g., transformation, transfection,electroporation, infection), such that the nucleic acid molecule(s) areoperably linked to one or more expression control elements (e.g., in avector, in a construct created by processes in the cell, integrated intothe host cell genome). Host cells can be maintained under conditionssuitable for expression (e.g., in the presence of inducer, suitablemedia supplemented with appropriate salts, growth factors, antibiotic,nutritional supplements, etc.), whereby the encoded polypeptide(s) areproduced. If desired, the encoded protein (e.g., humanized 1D9 antibody)can be isolated from, e.g., the host cells, medium, milk. This processencompasses expression in a host cell of a transgenic animal (see e.g.,WO 92/03918, GenPharm International, published Mar. 19, 1992).

[0111] Fusion proteins can be produced in which a humanizedimmunoglobulin or immunoglobulin chain is linked to a non-immunoglobulinmoiety (i.e., a moiety which does not occur in immunoglobulins as foundin nature) in an N-terminal location, C-terminal location or internal tothe fusion protein. For example, some embodiments can be produced by theinsertion of a nucleic acid encoding immunoglobulin sequences into asuitable expression vector, such as a pET vector (e.g., pET-15b,Novagen), a phage vector (e.g., pCANTAB 5 E, Pharmacia), or other vector(e.g., pRIT2T Protein A fusion vector, Pharmacia). The resultingconstruct can be introduced into a suitable host cell for expression.Upon expression, some fusion proteins can be isolated or purified from acell lysate by means of a suitable affinity matrix (see e.g., CurrentProtocols in Molecular Biology (Ausubel, F. M. et al., eds., Vol. 2,Suppl. 26, pp. 16.4.1-16.7.8 (1991)).

[0112] Therapeutic Methods and Compositions

[0113] The present invention provides humanized immunoglobulins which(1) can bind CCR2 in vitro and/or in vivo; and/or (2) can modulate anactivity or function of CCR2, such as (a) binding function (e.g., theability of CCR2 to bind to a ligand) and/or (b) leukocyte trafficking,including recruitment and/or accumulation of leukocytes in tissues.Preferably the humanized immunoglobulins are capable of selectivelybinding CCR2 in vitro and/or in vivo, and inhibiting CCR2-mediatedinteractions. In one embodiment, a humanized immunoglobulin can bindCCR2, and can inhibit binding of CCR2 to one or more of its ligands(e.g., HIV, MCP-1, MCP-2, MCP-3, MCP-4).

[0114] The humanized immunoglobulins of the present invention are usefulin a variety of processes with applications in research, diagnosis andtherapy. For instance, they can be used to detect, isolate, and/orpurify CCR2 or variants thereof (e.g., by affinity purification or othersuitable methods), and to study CCR2 structure (e.g., conformation) andfunction. The humanized immunoglobulins of the present invention canalso be used in diagnostic applications (e.g., in vitro, ex vivo) or tomodulate CCR2 function in therapeutic (including prophylactic)applications.

[0115] For example, the humanized immunoglobulins of the presentinvention can be used to detect and/or measure the level of CCR2 in asample (e.g., tissues or body fluids, such as an inflammatory exudate,blood, serum, bowel fluid, on cells bearing CCR2). For example, a sample(e.g., tissue and/or body fluid) can be obtained from an individual anda suitable immunological method can be used to detect and/or measureCCR2 expression, including methods such as enzyme-linked immunosorbentassays (ELISA), including chemiluminescence assays, radioimmunoassay,and immunohistology. In one embodiment, a method of detecting a selectedCCR2 in a sample is provided, comprising contacting a sample with ahumanized immunoglobulin of the present invention under conditionssuitable for specific binding of the humanized immunoglobulin to CCR2and detecting antibody-CCR2 complexes which are formed. In anapplication of the method, humanized immunoglobulins can be used toanalyze normal versus inflamed tissues (e.g., from a human) for CCR2reactivity and/or expression (e.g., immunohistologically)), to detectassociations between particular conditions and increased expression ofCCR2 (e.g., in affected tissues). The humanized immunoglobulins of thepresent invention permit immunological methods of assessment of thepresence of CCR2 in normal versus inflamed tissues, through which thepresence of disease, disease progress and/or the efficacy of anti-CCR2integrin therapy in inflammatory disease can be assessed.

[0116] The humanized immunoglobulins of the present invention can alsobe used to modulate (e.g., inhibit (reduce or prevent)) binding functionand/or leukocyte (e.g., lymphocyte, monocyte) trafficking modulated byCCR2. For example, humanized immunoglobulins which inhibit the bindingof CCR2 to a ligand (i.e., one or more ligands) can be administeredaccording to the method in the treatment of diseases associated withleukocyte (e.g., lymphocyte, monocyte) infiltration of tissues.Additionally, humanized immunoglobulins which inhibit the binding ofCCR2 to a ligand (i.e., one or more ligands) can be administeredaccording to the method in the treatment of HIV. An effective amount ofa humanized immunoglobulin of the present invention (i.e., one or more)is administered to an individual (e.g., a mammal, such as a human orother primate) in order to treat such a disease.

[0117] The humanized immunoglobulin is administered in an effectiveamount which inhibits binding of CCR2 to a ligand thereof. For therapy,an effective amount will be sufficient to achieve the desiredtherapeutic (including prophylactic) effect (such as an amountsufficient to reduce or prevent CCR2-mediated binding and/orsignalling). The humanized immunoglobulin can be administered in asingle dose or multiple doses. The dosage can be determined by methodsknown in the art and can be dependent, for example, upon theindividual's age, sensitivity, tolerance and overall well-being.Suitable dosages for antibodies can be from about 0.1 mg/kg body weightto about 10.0 mg/kg body weight per treatment.

[0118] According to the method, the humanized immunoglobulin can beadministered to an individual (e.g., a human) alone or in conjunctionwith another agent. A humanized immunoglobulin can be administeredbefore, along with or subsequent to administration of the additionalagent. Thus, the invention includes pharmaceutical compositionscomprising a humanized immunoglobulin or antigen-binding fragmentthereof of the invention and a suitable carrier. In one embodiment, morethan one humanized immunoglobulin which inhibits the binding of CCR2 toits ligands is administered. In another embodiment an additionalmonoclonal antibody is administered in addition to a humanizedimmunoglobulin of the present invention. In yet another embodiment, anadditional pharmacologically active ingredient (e.g., anantiinflammatory compound, such as sulfasalazine, another non-steroidalantiinflammatory compound, or a steroidal antiinflammatory compound) canbe administered in conjunction with a humanized immunoglobulin of thepresent invention.

[0119] A variety of routes of administration are possible, including,but not necessarily limited to, parenteral (e.g., intravenous,intraarterial, intramuscular, subcutaneous injection), oral (e.g.,dietary), topical, inhalation (e.g., intrabronchial, intranasal or oralinhalation, intranasal drops), or rectal, depending on the disease orcondition to be treated. Parenteral administration is a preferred modeof administration.

[0120] Formulation will vary according to the route of administrationselected (e.g., solution, emulsion). An appropriate compositioncomprising the humanized antibody to be administered can be prepared ina physiologically acceptable vehicle or carrier. For solutions oremulsions, suitable carriers include, for example, aqueous oralcoholic/aqueous solutions, emulsions or suspensions, including salineand buffered media. Parenteral vehicles can include sodium chloridesolution, Ringer's dextrose, dextrose and sodium chloride, lactatedRinger's or fixed oils. Intravenous vehicles can include variousadditives, preservatives, or fluid, nutrient or electrolyte replenishers(See, generally, Remington's Pharmaceutical Sciences, 17th Edition, MackPublishing Co., Pa., 1985). For inhalation, the compound can besolubilized and loaded into a suitable dispenser for administration(e.g., an atomizer, nebulizer or pressurized aerosol dispenser).

[0121] Thus, the invention includes a method of inhibiting HIV infectionof a cell, comprising contacting a cell with an effective amount of acomposition comprising a humanized immunoglobulin or antigen-bindingfragment thereof of the invention. The invention also relates to amethod of treating HIV or inhibiting HIV infection in a patientcomprising administering to the patient a composition comprising aneffective amount of a humanized immunoglobulin of or antigen-bindingfragment thereof of the invention.

[0122] The invention also relates to a method of inhibiting a functionassociated with binding of a chemokine to mammalian CCR2 or a functionalportion of CCR2, comprising contacting a composition comprising CCR2 orportion thereof with an effective amount of a humanized immunoglobulinor antigen-binding fragment thereof of the invention, wherein saidhumanized immunoglobulin inhibits binding of the chemokine to mammalianCCR2 and inhibits one or more functions associated with binding of thechemokine to CCR2. For example, the chemokine can be selected from thegroup consisting of MCP-1, MCP-2, MCP-3, MCP-4 and combinations thereof.

[0123] The invention also relates to a method of inhibiting leukocytetrafficking in a patient, comprising administering to the patient acomposition comprising an effective amount of a humanized immunoglobulinor antigen-binding fragment thereof of the invention which binds tomammalian CCR2 and inhibits binding of a ligand to the receptor. Forexample, the ligand can be a chemokine (e.g., MCP-1, MCP-2, MCP-3,MCP-4) or HIV.

[0124] The invention also relates to a method of inhibiting theinteraction of a first cell expressing CCR2 with a ligand (e.g., on asecond cell expressing a ligand of CCR2), comprising contacting thefirst cell with an effective amount of a humanized immunoglobulin orantigen-binding fragment thereof of the invention, particularly whereinsaid immunoglobulin or fragment inhibits the binding of ligand to CCR2.For example, the cell can be selected from the group consisting oflymphocytes, monocytes, granulocytes, T cells, basophils, and cellscomprising a recombinant nucleic acid encoding CCR2 or a portionthereof. In one embodiment, the ligand is a chemokine (e.g., MCP-1,MCP-2, MCP-3, MCP-4). In another embodiment, the ligand is HIV.

[0125] The invention also includes a method of treating a CCR2-mediateddisorder in a patient, comprising administering to the patient aneffective amount of a humanized immunoglobulin or antigen-bindingfragment thereof of the invention which binds to mammalian CCR2. Thedisorder can include, but is not limited to, allergy, atherogenesis,anaphylaxis, malignancy, chronic and acute inflammatory disorders,histamine and IgE-mediated allergic reactions, shock, and rheumatoidarthritis, atherosclerosis, multiple sclerosis, stenosis, restenosis,allograft rejection, fibrotic disease, asthma, and inflammatoryglomerulopathies.

[0126] In a particular embodiment, the invention relates to a method ofinhibiting restenosis in a patient, comprising administering to thepatient an effective amount of a humanized immunoglobulin orantigen-binding fragment thereof of the invention which binds tomammalian CCR2. The invention also includes a humanized immunoglobulinor antigen-binding fragment thereof of the invention for use in therapyor diagnosis or for use in treating a CCR2-mediated disease or disorder.The invention also includes the use of a humanized immunoglobulin orantigen-binding fragment thereof of the invention for the manufacture ofa medicament for treating a CCR2-mediated disease.

[0127] Anti-idiotypic antibodies are also provided. Anti-idiotypicantibodies recognize antigenic determinants associated with theantigen-binding site of another antibody. Anti-idiotypic antibodies canbe prepared against second antibody by immunizing an animal of the samespecies, and preferably of the same strain, as the animal used toproduce the second antibody. See e.g., U.S. Pat. No. 4,699,880.

[0128] The present invention also pertains to the hybridoma cell linesdeposited under ATCC Accession Nos. HB-12549 and HB-12550, as well as tothe monoclonal antibodies produced by the hybridoma cell lines depositedunder ATCC Accession Nos. HB-12549 and HB-12550. The cell lines of thepresent invention have uses other than for the production of themonoclonal antibodies. For example, the cell lines of the presentinvention can be fused with other cells (such as suitably drug-markedhuman myeloma, mouse myeloma, human-mouse heteromyeloma or humanlymphoblastoid cells) to produce additional hybridomas, and thus providefor the transfer of the genes encoding the monoclonal antibodies. Inaddition, the cell lines can be used as a source of nucleic acidsencoding the anti-CCR2 immunoglobulin chains, which can be isolated andexpressed (e.g., upon transfer to other cells using any suitabletechnique (see e.g., Cabilly et al., U.S. Pat. No. 4,816,567; Winter,U.S. Pat. No. 5,225,539)). For instance, clones comprising a rearrangedanti-CCR2 light or heavy chain can be isolated (e.g., by PCR) or cDNAlibraries can be prepared from mRNA isolated from the cell lines, andcDNA clones encoding an anti-CCR2 immunoglobulin chain can be isolated.Thus, nucleic acids encoding the heavy and/or light chains of theantibodies or portions thereof can be obtained and used in accordancewith recombinant DNA techniques for the production of the specificimmunoglobulin, immunoglobulin chain, or variants thereof (e.g.,humanized immunoglobulins) in a variety of host cells or in an in vitrotranslation system. For example, the nucleic acids, including cDNAs, orderivatives thereof encoding variants such as a humanized immunoglobulinor immunoglobulin chain, can be placed into suitable prokaryotic oreukaryotic vectors (e.g., expression vectors) and introduced into asuitable host cell by an appropriate method (e.g., transformation,transfection, electroporation, infection), such that the nucleic acid isoperably linked to one or more expression control elements (e.g., in thevector or integrated into the host cell genome). For production, hostcells can be maintained under conditions suitable for expression (e.g.,in the presence of inducer, suitable media supplemented with appropriatesalts, growth factors, antibiotic, nutritional supplements, etc.),whereby the encoded polypeptide is produced. If desired, the encodedprotein can be recovered and/or isolated (e.g., from the host cells,medium, milk). It will be appreciated that the method of productionencompasses expression in a host cell of a transgenic animal (see e.g.,WO 92/03918, GenPharm International, published Mar. 19, 1992).

[0129] As described herein, antibodies and functional fragments thereofof the present invention can block (inhibit) binding of a ligand to CCR2and/or inhibit function associated with binding of the ligand to theCCR2. As discussed below various methods can be used to assessinhibition of binding of a ligand to CCR2 and/or function associatedwith binding of the ligand to the receptor.

[0130] Binding Assays

[0131] As used herein “mammalian CCR2 protein” refers to naturallyoccurring or endogenous mammalian CCR2 proteins and to proteins havingan amino acid sequence which is the same as that of a naturallyoccurring or endogenous corresponding mammalian CCR2 protein (e.g.,recombinant proteins). Accordingly, as defined herein, the term includesmature receptor protein, polymorphic or allelic variants, and otherisoforms of a mammalian CCR2 (e.g., produced by alternative splicing orother cellular processes), and modified or unmodified forms of theforegoing (e.g., glycosylated, unglycosylated). Mammalian CCR2 proteinscan be isolated and/or recombinant proteins (including syntheticallyproduced proteins). Naturally occurring or endogenous mammalian CCR2proteins include wild type proteins such as mature CCR2, polymorphic orallelic variants and other isoforms which occur naturally in mammals(e.g., humans, non-human primates), such as the CCR2a and CCR2b forms ofthe receptor protein which are produced by alternative splicing of thecarboxy-terminus of the protein. Such proteins can be recovered orisolated from a source which naturally produces mammalian CCR2, forexample. These proteins and mammalian CCR2 proteins having the sameamino acid sequence as a naturally occurring or endogenous correspondingmammalian CCR2, are referred to by the name of the corresponding mammal.For example, where the corresponding mammal is a human, the protein isdesignated as a human CCR2 protein (e.g., a recombinant human CCR2produced in a suitable host cell).

[0132] “Functional variants” of mammalian CCR2 proteins includefunctional fragments, functional mutant proteins, and/or functionalfusion proteins (e.g., produced via mutagenesis and/or recombinanttechniques). Generally, fragments or portions of mammalian CCR2 proteinsinclude those having a deletion (i.e., one or more deletions) of anamino acid (i.e., one or more amino acids) relative to the maturemammalian CCR2 protein (such as N-terminal, C-terminal or internaldeletions). Fragments or portions in which only contiguous amino acidshave been deleted or in which non-contiguous amino acids have beendeleted relative to mature mammalian CCR2 protein are also envisioned.

[0133] Generally, mutants of mammalian CCR2 proteins include natural orartificial variants of a mammalian CCR2 protein differing by theaddition, deletion and/or substitution of one or more contiguous ornon-contiguous amino acid residues (e.g., receptor chimeras). Suchmutations can be in a conserved region or nonconserved region (comparedto other CXC and/or CC chemokine receptors), extracellular, cytoplasmic,or transmembrane region, for example.

[0134] Generally, fusion proteins encompass polypeptides comprising amammalian CCR2 (e.g., human CCR2) as a first moiety, linked via apeptide cond to a second moiety not occurring in the mammalian CCR2 asfound in nature. Thus, the second moiety can be an amino acid,oligopeptide or polypeptide. The first moiety can be in an N-terminallocation, C-terminal location or internal to the fusion protein. In oneembodiment, the fusion protein comprises an affinity ligand (e.g., anenzyme, an antigen, epitope tage) as the first moiety, and a secondmoiety comprising a linker sequence and human CCR2 or a portion thereof.

[0135] A “functional fragment or portion”, “functional mutant” and/or“functional fusion protein” of a mammalian CCR2 protein refers to anisolated and/or recombinant protein or polypeptide which has at leastone function characteristic of a mammalian CCR2 protein as describedherein, such as a binding activity, a signaling activity and/or abilityto stimulate a cellular response. Preferred functional variants can binda ligand (i.e., one or more ligands such as MCP-1, MCP-2, MCP-3 and/orMCP-4), and are referred to herein as “ligand binding variants”.

[0136] In one embodiment, a functional variant of mammalian CCR2 sharesat least about 85% sequence identity with said mammalian CCR2,preferably at least about 90% sequence identity, and more preferably atleast about 95% sequence identity with said mammalian CCR2. The nucleicacid and amino acid sequences of human CCR2a and CCR2b are described inU.S. Pat. No. 5,707,815. Sequence identity can be determine using asuitable program, such as the Blastx program (Version 1.4), usingappropriate parameters, such as default parameters. In one embodiment,parameters for Blastx search are scoring matrix BLOSUM62, W=3. Inanother embodiment, a functional variant comprises a nucleic acidsequence which is different from the naturally-occurring nucleic acidmolecule but which, due to the degeneracy of the genetic code, encodesmammalian CCR2 or a portion thereof.

[0137] A composition comprising an isolated and/or recombinant mammalianCCR2 or functional variant thereof can be maintained under conditionssuitable for binding, the mammalian CCR2 or variant is contacted with anantibody or fragment to be tested, and binding is detected or measureddirectly or indirectly. In one embodiment, cells which naturally expressCCR2 or cells comprising a recombinant nucleic acid sequence whichencodes a mammalian CCR2 or variant thereof are used. The cells aremaintained under conditions appropriate for expression of receptor. Thecells are contacted with an antibody or fragment under conditionssuitable for binding (e.g., in a suitable binding buffer), and bindingis detected by standard techniques. To determine binding, the extent ofbinding can be determined relative to a suitable control (e.g., comparedwith background determined in the absence of antibody, compared withbinding of a second antibody (i.e., a standard), compared with bindingof antibody to untransfected cells). A cellular fraction, such as amembrane fraction, containing receptor or liposomes comprising receptorcan be used in lieu of whole cells.

[0138] In one embodiment, the antibody is labeled with a suitable label(e.g., fluorescent label, isotope label, antigen or epitope label,enzyme label), and binding is determined by detection of the label. Inanother embodiment, bound antibody can be detected by labeled secondantibody. Specificity of binding can be assessed by competition ordisplacement, for example, using unlabeled antibody or a ligand ascompetitor.

[0139] Binding inhibition assays can also be used to identify antibodiesor fragments thereof which bind CCR2 and inhibit binding of anothercompound such as a ligand (e.g., MCP-1, MCP-2, MCP-3 and/or MCP-4) toCCR2 or a functional variant. For example, a binding assay can beconducted in which a reduction in the binding of a ligand of CCR2 (inthe presence of an antibody), as compared to binding of the ligand inthe absence of the antibody, is detected or measured. A compositioncomprising an isolated and/or recombinant mammalian CCR2 or functionalvariant thereof can be contacted with the ligand and antibodysimultaneously, or one after the other, in either order. A reduction inthe extent of binding of the ligand in the presence of the antibody, isindicative of inhibition of binding by the antibody. For example,binding of the ligand could be decreased or abolished.

[0140] In one embodiment, direct inhibition of the binding of a ligand(e.g., a chemokine such as MCP-1) to a mammalian CCR2 or variant thereofby an antibody or fragment is monitored. For example, the ability of anantibody to inhibit the binding of ¹²⁵I-labeled MCP-1, ¹²⁵I-labeledMCP-2, ¹²⁵I-labeled MCP-3 or ²⁵I-labeled MCP-4 to mammalian CCR2 can bemonitored. Such an assay can be conducted using suitable cells bearingCCR2 or a functional variant thereof, such as isolated blood cells(e.g., T cells, PBMC) or a suitable cell line naturally expressing CCR2,or a cell line containing nucleic acid encoding a mammalian CCR2, or amembrane fraction from said cells, for instance.

[0141] Other methods of identifying the presence of an antibody whichbinds CCR2 are available, such as other suitable binding assays, ormethods which monitor events which are triggered by receptor binding,including signaling function and/or stimulation of a cellular response(e.g., leukocyte trafficking).

[0142] It will be understood that the inhibitory effect of antibodies ofthe present invention can be assessed in a binding inhibition assay.Competition between antibodies for receptor binding can also be assessedin the method. Antibodies which are identified in this manner can befurther assessed to determine whether, subsequent to binding, they actto inhibit other functions of CCR2 and/or to assess their therapeuticutility.

[0143] Signaling Assays

[0144] The binding of a ligand or promoter, such as an agonist, to CCR2can result in signaling by this G protein-coupled receptor, and theactivity of G proteins as well as other intracellular signalingmolecules is stimulated. The induction of signaling function by acompound (e.g., an antibody or fragment thereof) can be monitored usingany suitable method. Such an assay can be used to identify antibodyagonists of CCR2. The inhibitory activity of an antibody or functionalfragment thereof can be determined using a ligand or promoter in theassay, and assessing the ability of the antibody to inhibit the activityinduced by ligand or promoter.

[0145] G protein activity, such as hydrolysis of GTP to GDP, or latersignaling events triggered by receptor binding, such as induction ofrapid and transient increase in the concentration of intracellular(cytosolic) free calcium [Ca²⁺]i, can be assayed by methods known in theart or other suitable methods (see e.g., Neote, K. et al., Cell, 72:415-425 1993); Van Riper et al., J. Exp. Med., 177: 851-856 (1993);Dahinden, C. A. et al., J. Exp. Med., 179: 751-756 (1994)).

[0146] For example, the functional assay of Sledziewski et al. usinghybrid G protein coupled receptors can be used to monitor the ability aligand or promoter to bind receptor and activate a G protein(Sledziewski et al., U.S. Pat. No. 5,284,746, the teachings of which areincorporated herein by reference).

[0147] Such assays can be performed in the presence of the antibody orfragment thereof to be assessed, and the ability of the antibody orfragment to inhibit the activity induced by the ligand or promoter isdetermined using known methods and/or methods described herein.

[0148] Chemotaxis and Assays of Cellular Stimulation

[0149] Chemotaxis assays can also be used to assess the ability of anantibody or functional fragment thereof to block binding of a ligand tomammalian CCR2 or functional variant thereof and/or inhibit functionassociated with binding of the ligand to the receptor. These assays arebased on the functional migration of cells in vitro or in vivo inducedby a compound. Chemotaxis can be assessed as described in the Examples,e.g., in an assay utilizing a 96-well chemotaxis plate, or using otherart-recognized methods for assessing chemotaxis. For example, the use ofan in vitro transendothelial chemotaxis assay is described by Springeret al. (Springer et al., WO 94/20142, published Sep. 15, 1994, theteachings of which are incorporated herein by reference; see also Bermanet al., Immunol. Invest. 17: 625-677 (1988)). Migration acrossendothelium into collagen gels has also been described (Kavanaugh etal., J. Immunol., 146: 4149-4156 (1991)). Stable transfectants of mouseL1-2 pre-B cells or of other suitable host cells capable of chemotaxiscan be used in chemotaxis assays, for example.

[0150] Generally, chemotaxis assays monitor the directional movement ormigration of a suitable cell (such as a leukocyte (e.g., lymphocyte,eosinophil, basophil)) into or through a barrier (e.g., endothelium, afilter), toward increased levels of a compound, from a first surface ofthe barrier toward an opposite second surface. Membranes or filtersprovide convenient barriers, such that the directional movement ormigration of a suitable cell into or through a filter, toward increasedlevels of a compound, from a first surface of the filter toward anopposite second surface of the filter, is monitored. In some assays, themembrane is coated with a substance to facilitate adhesion, such asICAM-1, fibronectin or collagen. Such assays provide an in vitroapproximation of leukocyte “homing”.

[0151] For example, one can detect or measure inhibition of themigration of cells in a suitable container (a containing means), from afirst chamber into or through a microporous membrane into a secondchamber which contains an antibody to be tested, and which is dividedfrom the first chamber by the membrane. A suitable membrane, having asuitable pore size for monitoring specific migration in response tocompound, including, for example, nitrocellulose, polycarbonate, isselected. For example, pore sizes of about 3-8 microns, and preferablyabout 5-8 microns can be used. Pore size can be uniform on a filter orwithin a range of suitable pore sizes.

[0152] To assess migration and inhibition of migration, the distance ofmigration into the filter, the number of cells crossing the filter thatremain adherent to the second surface of the filter, and/or the numberof cells that accumulate in the second chamber can be determined usingstandard techniques (e.g., microscopy). In one embodiment, the cells arelabeled with a detectable label (e.g., radioisotope, fluorescent label,antigen or epitope label), and migration can be assessed in the presenceand absence of the antibody or fragment by determining the presence ofthe label adherent to the membrane and/or present in the second chamberusing an appropriate method (e.g., by detecting radioactivity,fluorescence, immunoassay). The extent of migration induced by anantibody agonist can be determined relative to a suitable control (e.g.,compared to background migration determined in the absence of theantibody, compared to the extent of migration induced by a secondcompound (i.e., a standard), compared with migration of untransfectedcells induced by the antibody).

[0153] In one embodiment, particularly for T cells, monocytes or cellsexpressing a mammalian CCR2, transendothelial migration can bemonitored. In this embodiment, transmigration through an endothelialcell layer is assessed. To prepare the cell layer, endothelial cells canbe cultured on a microporous filter or membrane, optionally coated witha substance such as collagen, fibronectin, or other extracellular matrixproteins, to facilitate the attachment of endothelial cells. Preferably,endothelial cells are cultured until a confluent monolayer is formed. Avariety of mammalian endothelial cells can are available for monolayerformation, including for example, vein, artery or microvascularendothelium, such as human umbilical vein endothelial cells (CloneticsCorp, San Diego, Calif.). To assay chemotaxis in response to aparticular mammalian receptor, endothelial cells of the same mammal arepreferred; however endothelial cells from a heterologous mammalianspecies or genus can also be used.

[0154] Generally, the assay is performed by detecting the directionalmigration of cells into or through a membrane or filter, in a directiontoward increased levels of a compound, from a first surface of thefilter toward an opposite second surface of the filter, wherein thefilter contains an endothelial cell layer on a first surface.Directional migration occurs from the area adjacent to the firstsurface, into or through the membrane, towards a compound situated onthe opposite side of the filter. The concentration of compound presentin the area adjacent to the second surface, is greater than that in thearea adjacent to the first surface.

[0155] In one embodiment used to test for an antibody inhibitor, acomposition comprising cells capable of migration and expressing amammalian CCR2 receptor can be placed in the first chamber. Acomposition comprising one or more ligands or promoters capable ofinducing chemotaxis of the cells in the first chamber (havingchemoattractant function) is placed in the second chamber. Preferablyshortly before the cells are placed in the first chamber, orsimultaneously with the cells, a composition comprising the antibody tobe tested is placed, preferably, in the first chamber. Antibodies orfunctional fragments thereof which can bind receptor and inhibit theinduction of chemotaxis, by a ligand or promoter, of the cellsexpressing a mammalian CCR2 in this assay are inhibitors of receptorfunction (e.g., inhibitors of stimulatory function). A reduction in theextent of migration induced by the ligand or promoter in the presence ofthe antibody or fragment is indicative of inhibitory activity. Separatebinding studies (see above) could be performed to determine whetherinhibition is a result of binding of the antibody to receptor or occursvia a different mechanism.

[0156] In vivo assays which monitor leukocyte infiltration of a tissue,in response to injection of a compound (e.g., chemokine or antibody) inthe tissue, are described below (see Models of Inflammation). Thesemodels of in vivo homing measure the ability of cells to respond to aligand or promoter by emigration and chemotaxis to a site ofinflammation and to assess the ability of an antibody or fragmentthereof to block this emigration.

[0157] In addition to the methods described, the effects of an antibodyor fragment on the stimulatory function of CCR2 can be assessed bymonitoring cellular responses induced by active receptor, using suitablehost cells containing receptor.

[0158] Identification of Additional Ligands, Inhibitors and/or Promotersof Mammalian CCR2 Function

[0159] The assays described above, which can be used to assess bindingand function of the antibodies and fragments of the present invention,can be adapted to identify additional ligands or other substances whichbind a mammalian CCR2 or functional variant thereof, as well asinhibitors and/or promoters of mammalian CCR2 function. For example,agents having the same or a similar binding specificity as that of anantibody of the present invention or functional portion thereof can beidentified by a competition assay with said antibody or portion thereof.Thus, the present invention also encompasses methods of identifyingligands of the receptor or other substances which bind a mammalian CCR2protein, as well as inhibitors (e.g., antagonists) or promoters (e.g.,agonists) of receptor function. In one embodiment, cells bearing amammalian CCR2 protein or functional variant thereof (e.g., leukocytes,cell lines or suitable host cells which have been engineered to expressa mammalian CCR2 protein or functional variant encoded by a nucleic acidintroduced into said cells) are used in an assay to identify and assessthe efficacy of ligands or other substances which bind receptor,including inhibitors or promoters of receptor function. Such cells arealso useful in assessing the function of the expressed receptor proteinor polypeptide.

[0160] According to the present invention, ligands and other substanceswhich bind receptor, inhibitors and promoters of receptor function canbe identified in a suitable assay, and further assessed for therapeuticeffect. Inhibitors of receptor function can be used to inhibit (reduceor prevent) receptor activity, and ligands and/or promoters can be usedto induce (trigger or enhance) normal receptor function where indicated.Thus, the present invention provides a method of treating inflammatorydiseases, including autoimmune disease and graft rejection, comprisingadministering an inhibitor of receptor function to an individual (e.g.,a mammal). The present invention further provides a method ofstimulating receptor function by administering a novel ligand orpromoter of receptor function to an individual, providing a new approachto selective stimulation of leukocyte function, which is useful, forexample, in the treatment of infectious diseases and cancer.

[0161] As used herein, a “ligand” of a mammalian CCR2 protein refers toa particular class of substances which bind to a mammalian CCR2 protein,including natural ligands and synthetic and/or recombinant forms ofnatural ligands. Infectious agents having a tropism for mammalianCCR2-positive cells (e.g., viruses such as HIV) can also bind to amammalian CCR2 protein. A natural ligand of a selected mammalianreceptor is of a mammalian origin which is the same as that of themammalian CCR2 protein (e.g., a chemokine such as MCP-1, MCP-2, MCP-3and/or MCP-4). In a preferred embodiment, ligand binding of a mammalianCCR2 protein occurs with high affinity.

[0162] As used herein, an “inhibitor” is a substance which inhibits(decreases or prevents) at least one function characteristic of amammalian CCR2 protein (e.g., a human CCR2), such as a binding activity(e.g., ligand binding, promoter binding, antibody binding), a signalingactivity (e.g., activation of a mammalian G protein, induction of rapidand transient increase in the concentration of cytosolic free calcium[Ca²⁺]i), and/or cellular response function (e.g., stimulation ofchemotaxis, exocytosis or inflammatory mediator release by leukocytes).An inhibitor is also a substance which inhibits HIV entry into a cell.The term inhibitor refers to substances including antagonists which bindreceptor (e.g., an antibody, a mutant of a natural ligand, smallmolecular weight organic molecules, other competitive inhibitors ofligand binding), and substances which inhibit receptor function withoutbinding thereto (e.g., an anti-idiotypic antibody).

[0163] As used herein, a “promoter” is a substance which promotes(induces, causes, enhances or increases) at least one functioncharacteristic of a mammalian CCR2 protein (e.g., a human CCR2), such asa binding activity (e.g., ligand, inhibitor and/or promoter binding), asignaling activity (e.g., activation of a mammalian G protein, inductionof rapid and transient increase in the concentration of cytosolic freecalcium [Ca²⁺]i), and/or a cellular response function (e.g., stimulationof chemotaxis, exocytosis or inflammatory mediator release byleukocytes). The term promoter refers to substances including agonistswhich bind receptor (e.g., an antibody, a homolog of a natural ligandfrom another species), and substances which promote receptor functionwithout binding thereto (e.g., by activating an associated protein). Ina preferred embodiment, the agonist is other than a homolog of a naturalligand.

[0164] Thus, the invention also relates to a method of detecting oridentifying an agent which binds a mammalian CC-chemokine receptor 2 orligand binding variant thereof, including ligands, inhibitors,promoters, and other substances which bind a mammalian CCR2 receptor orfunctional variant. According to the method, an agent to be tested, anantibody or antigen-binding fragment of the present invention (e.g.,8G2, 1 D9, an antibody having an epitopic specificity which is the sameas or similar to that of 8G2 or 1D9, and antigen-binding fragmentsthereof) and a composition comprising a mammalian CC-chemokine receptor2 or a ligand binding variant thereof can be combined. The foregoingcomponents are combined under conditions suitable for binding of theantibody or antigen-binding fragment to mammalian CC-chemokine receptor2 or a ligand binding variant thereof, and binding of the antibody orfragment to the mammalian CC-chemokine receptor 2 or ligand bindingvariant is detected or measured, either directly or indirectly,according to methods described herein or other suitable methods. Adecrease in the amount of complex formed relative to a suitable control(e.g., in the absence of the agent to be tested) is indicative that theagent binds said receptor or variant. The composition comprising amammalian CC-chemokine receptor 2 or a ligand binding variant thereofcan be a membrane fraction of a cell bearing recombinant chemokinereceptor 2 protein or ligand binding variant thereof. The antibody orfragment thereof can be labeled with a label such as a radioisotope,spin label, antigen or epitope label, enzyme label, fluorescent groupand chemiluminescent group.

[0165] In one embodiment, the invention relates to a method of detectingor identifying an agent which binds a mammalian CC-chemokine receptor 2or a ligand binding variant thereof, comprising combining an agent to betested, an antibody or antigen-binding fragment of the present invention(e.g., 1D9, 8G2, an antibody having an epitopic specificity which is thesame as or similar to that of 1D9 or 8G2, or antigen-binding fragmentsthereof) and a cell bearing a mammalian CC-chemokine receptor 2 or aligand binding variant thereof. The foregoing components are combinedunder conditions suitable for binding of the antibody or antigen-bindingfragment to the CCR2 protein or ligand binding variant thereof, andbinding of the antibody or fragment to the mammalian CC-chemokinereceptor 2 or variant is detected or measured, either directly orindirectly, by methods described herein and or other suitable methods. Adecrease in the amount of complex formed relative to a suitable controlis indicative that the agent binds the receptor or variant. The antibodyor fragment thereof can be labeled with a label selected from the groupconsisting of a radioisotope, spin label, antigen or epitope label,enzyme label, fluorescent group and chemiluminescent group. These andsimilar assays can be used to detect agents, including ligands (e.g.,chemokines or strains of HIV which interact with CCR2) or othersubstances, including inhibitors or promoters of receptor function,which can bind CCR2 and compete with the antibodies described herein forbinding to the receptor.

[0166] The assays described above can be used, alone or in combinationwith each other or other suitable methods, to identify ligands or othersubstances which bind a mammalian CCR2 protein, and inhibitors orpromoters of a mammalian CCR2 protein or variant. The in vitro methodsof the present invention can be adapted for high-throughput screening inwhich large numbers of samples are processed (e.g., a 96-well format).Cells expressing mammalian CCR2 (e.g., human CCR2) at levels suitablefor high-throughput screening can be used, and thus, are particularlyvaluable in the identification and/or isolation of ligands or othersubstances which bind receptor, and inhibitors or promoters of mammalianCCR2 proteins. Expression of receptor can be monitored in a variety ofways. For instance, expression can be monitored using antibodies of thepresent invention which bind receptor or a portion thereof. Also,commercially available antibodies can be used to detect expression of anantigen- or epitope-tagged fusion protein comprising a receptor proteinor polypeptide (e.g., FLAG tagged receptors), and cells expressing thedesired level can be selected.

[0167] Nucleic acid encoding a mammalian CCR2 protein or functionalvariant thereof can be incorporated into an expression system to producea receptor protein or polypeptide. An isolated and/or recombinantmammalian CCR2 protein or variant, such as a receptor expressed in cellsstably or transiently transfected with a construct comprising arecombinant nucleic acid encoding a mammalian CCR2 protein or variant,or in a cell fraction containing receptor (e.g., a membrane fractionfrom transfected cells, liposomes incorporating receptor), can be usedin tests for receptor function. The receptor can be further purified ifdesired. Testing of receptor function can be carried out in vitro or invivo.

[0168] An isolated and/or recombinant mammalian CCR2 protein orfunctional variant thereof, such as a human CCR2, can be used in thepresent method, in which the effect of a compound is assessed bymonitoring receptor function as described herein or using other suitabletechniques. For example, stable or transient transfectants (e.g.,baculovirus infected Sf9 cells, stable tranfectants of mouse L1/2 pre-Bcells), can be used in binding assays. Stable transfectants of Jurkatcells or of other suitable cells capable of chemotaxis can be used(e.g., mouse L1/2 pre-B cells) in chemotaxis assays, for example.

[0169] According to the method of the present invention, compounds canbe individually screened or one or more compounds can be testedsimultaneously according to the methods herein. Where a mixture ofcompounds is tested, the compounds selected by the processes describedcan be separated (as appropriate) and identified by suitable methods(e.g., PCR, sequencing, chromatography, mass spectroscopy). The presenceof one or more compounds (e.g., a ligand, inhibitor, promoter) in a testsample can also be determined according to these methods.

[0170] Large combinatorial libraries of compounds (e.g., organiccompounds, recombinant or synthetic peptides, “peptoids”, nucleic acids)produced by combinatorial chemical synthesis or other methods can betested (see e.g., Zuckerman, R. N. et al., J. Med. Chem., 37: 2678-2685(1994) and references cited therein; see also, Ohlmeyer, M. H. J. etal., Proc. Natl. Acad. Sci. USA 90:10922-10926 (1993) and DeWitt, S. H.et al., Proc. Natl. Acad. Sci. USA 90:6909-6913 (1993), relating totagged compounds; Rutter, W. J. et al. U.S. Pat. No. 5,010,175; Huebner,V. D. et al., U.S. Pat. No. 5,182,366; and Geysen, H. M., U.S. Pat. No.4,833,092). Where compounds selected from a combinatorial library by thepresent method carry unique tags, identification of individual compoundsby chromatographic methods is possible.

[0171] In one embodiment, phage display methodology is used. Forexample, a mammalian CCR2 protein or functional variant, an antibody orfunctional portion thereof of the present invention, and a phage (e.g.,a phage or collection of phage such as a library) displaying apolypeptide, can be combined under conditions appropriate for binding ofthe antibody or portion thereof to the mammalian CCR2 protein or variant(e.g., in a suitable binding buffer). Phage which can compete with theantibody or portion thereof and bind to the mammalian CCR2 protein orvariant can be detected or selected using standard techniques or othersuitable methods. Bound phage can be separated from receptor using asuitable elution buffer. For example, a change in the ionic strength orpH can lead to a release of phage. Alternatively, the elution buffer cancomprise a release component or components designed to disrupt bindingof compounds (e.g., one or more compounds which can disrupt binding ofthe displayed peptide to the receptor, such as a ligand, inhibitor,and/or promoter which competitively inhibits binding). Optionally, theselection process can be repeated or another selection step can be usedto further enrich for phage which bind receptor. The displayedpolypeptide can be characterized (e.g., by sequencing phage DNA). Thepolypeptides identified can be produced and further tested for binding,and for inhibitor or promoter function. Analogs of such peptides can beproduced which will have increased stability or other desirableproperties.

[0172] In one embodiment, phage expressing and displaying fusionproteins comprising a coat protein with an N-terminal peptide encoded byrandom sequence nucleic acids can be produced. Suitable host cellsexpressing a mammalian CCR2 protein or variant and an anti-CCR2 antibodyor functional portion thereof, are combined with the phage, bound phageare selected, recovered and characterized. (See e.g., Doorbar, J. and G.Winter, J. Mol. Biol., 244: 361 (1994) discussing a phage displayprocedure used with a G protein-coupled receptor).

[0173] Other sources of potential ligands or other substances which bindto, or inhibitors and/or promoters of, mammalian CCR2 proteins include,but are not limited to, variants of CCR2 ligands, including naturallyoccurring, synthetic or recombinant variants of MCP-1, MCP-2, MCP-3and/or MCP-4, substances such as other chemoattractants or chemokines,variants thereof, low molecular weight organic molecules, otherinhibitors and/or promoters (e.g., anti-CCR2 antibodies, antagonists,agonists), other G protein-coupled receptor ligands, inhibitors and/orpromoters (e.g., antagonists or agonists), and soluble portions of amammalian CCR2 receptor, such as a suitable receptor peptide or analogwhich can inhibit receptor function (see e.g., Murphy, R. B., WO94/05695).

[0174] Models of Inflammation

[0175] In vivo models of inflammation are available which can be used toassess the effects of antibodies and fragments of the invention in vivoas therapeutic agents. For example, leukocyte infiltration uponintradermal injection of a chemokine and an antibody or fragment thereofreactive with mammalian CCR2 into a suitable animal, such as rabbit,mouse, rat, guinea pig or rhesus macaque can be monitored (see e.g., VanDamme, J. et al., J. Exp. Med., 176: 59-65 (1992); Zachariae, C. O. C.et al., J. Exp. Med. 171: 2177-2182 (1990); Jose, P. J. et al., J. Exp.Med. 179: 881-887 (1994)). In one embodiment, skin biopsies are assessedhistologically for infiltration of leukocytes (e.g., eosinophils,granulocytes). In another embodiment, labeled cells (e.g., stablytransfected cells expressing a mammalian CCR2, labeled with ¹¹¹In forexample) capable of chemotaxis and extravasation are administered to theanimal. For example, an antibody or fragment to be assessed can beadministered, either before, simultaneously with or after ligand oragonist is administered to the test animal. A decrease of the extent ofinfiltration in the presence of antibody as compared with the extent ofinfiltration in the absence of inhibitor is indicative of inhibition.

[0176] Diagnostic and Therapeutic Applications

[0177] The antibodies and fragments of the present invention are usefulin a variety of applications, including research, diagnostic andtherapeutic applications. In one embodiment, the antibodies are labeledwith a suitable label (e.g., fluorescent label, chemiluminescent label,isotope label, antigen or epitope label or enzyme label). For instance,they can be used to isolate and/or purify receptor or portions thereof,and to study receptor structure (e.g., conformation) and function.

[0178] In addition, the various antibodies of the present invention canbe used to detect CCR2 or to measure the expression of receptor, forexample, on T cells (e.g., CD8+ cells, CD45RO+ cells), monocytes and/oron cells transfected with a receptor gene. Thus, they also have utilityin applications such as cell sorting (e.g., flow cytometry, fluorescenceactivated cell sorting), for diagnostic or research purposes.

[0179] The anti-CCR2 antibodies of the present invention have value indiagnostic applications. An anti-CCR2 antibody or fragment thereof canbe used to monitor expression of this receptor in HIV infectedindividuals, similar to the way anti-CD4 has been used as a diagnosticindicator of disease stage.

[0180] Typically, diagnostic assays entail detecting the formation of acomplex resulting from the binding of an antibody or fragment thereof toCCR2. For diagnostic purposes, the antibodies or antigen-bindingfragments can be labeled or unlabeled. The antibodies or fragments canbe directly labeled. A variety of labels can be employed, including, butnot limited to, radionuclides, fluorescers, enzymes, enzyme substrates,enzyme cofactors, enzyme inhibitors and ligands (e.g., biotin, haptens).Numerous appropriate immunoassays are known to the skilled artisan (see,for example, U.S. Pat. Nos. 3,817,827; 3,850,752; 3,901,654 and4,098,876). When unlabeled, the antibodies or fragments can be detectedusing suitable means, as in agglutination assays, for example. Unlabeledantibodies or fragments can also be used in combination with another(i.e., one or more) suitable reagent which can be used to detectantibody, such as a labeled antibody (e.g., a second antibody) reactivewith the first antibody (e.g., anti-idiotype antibodies or otherantibodies that are specific for the unlabeled immunoglobulin) or othersuitable reagent (e.g., labeled protein A).

[0181] In one embodiment, the antibodies or fragments of the presentinvention can be utilized in enzyme immunoassays, wherein the subjectantibody or fragment, or second antibodies, are conjugated to an enzyme.When a biological sample comprising a mammalian CCR2 protein is combinedwith the subject antibodies, binding occurs between the antibodies andCCR2 protein. In one embodiment, a sample containing cells expressing amammalian CCR2 protein, such as human blood, is combined with thesubject antibodies, and binding occurs between the antibodies and cellsbearing a human CCR2 protein comprising an epitope recognized by theantibody. These bound cells can be separated from unbound reagents andthe presence of the antibody-enzyme conjugate specifically bound to thecells can be determined, for example, by contacting the sample with asubstrate of the enzyme which produces a color or other detectablechange when acted on by the enzyme. In another embodiment, the subjectantibodies can be unlabeled, and a second, labeled antibody can be addedwhich recognizes the subject antibody.

[0182] Kits for use in detecting the presence of a mammalian CCR2protein in a biological sample can also be prepared. Such kits willinclude an antibody or functional fragment thereof which binds to amammalian CC-chemokine receptor 2 or portion of said receptor, as wellas one or more ancillary reagents suitable for detecting the presence ofa complex between the antibody or fragment and CCR2 or portion thereof.The antibody compositions of the present invention can be provided inlyophilized form, either alone or in combination with additionalantibodies specific for other epitopes. The antibodies, which can belabeled or unlabeled, can be included in the kits with adjunctingredients (e.g., buffers, such as Tris, phosphate and carbonate,stabilizers, excipients, biocides and/or inert proteins, e.g., bovineserum albumin). For example, the antibodies can be provided as alyophilized mixture with the adjunct ingredients, or the adjunctingredients can be separately provided for combination by the user.Generally these adjunct materials will be present in less than about 5%weight based on the amount of active antibody, and usually will bepresent in a total amount of at least about 0.001% weight based onantibody concentration. Where a second antibody capable of binding tothe monoclonal antibody is employed, such antibody can be provided inthe kit, for instance in a separate vial or container. The secondantibody, if present, is typically labeled, and can be formulated in ananalogous manner with the antibody formulations described above.

[0183] Similarly, the present invention also relates to a method ofdetecting and/or quantitating expression of a mammalian CCR2 or aportion of the receptor by a cell, in which a composition comprising acell or fraction thereof (e.g., membrane fraction) is contacted with anantibody or functional fragment thereof (e.g., 1D9 and/or 8G2) whichbinds to a mammalian CCR2 or portion of the receptor under conditionsappropriate for binding of the antibody or fragment thereto, and bindingis monitored. Detection of the antibody, indicative of the formation ofa complex between antibody and CCR2 or a portion thereof, indicates thepresence of the receptor. Binding of antibody to the cell can bedetermined as described above under the heading “Binding Assays”, forexample. The method can be used to detect expression of CCR2 on cellsfrom an individual (e.g., in a sample, such as a body fluid, such asblood, saliva or other suitable sample). The level of expression of CCR2on the surface of T cells or monocytes can also be determined, forinstance, by flow cytometry, and the level of expression (e.g., stainingintensity) can be correlated with disease susceptibility, progression orrisk.

[0184] Chemokine receptors function in the migration of leukocytesthroughout the body, particularly to inflammatory sites. Inflammatorycell emigration from the vasculature is regulated by a three-stepprocess involving interactions of leukocyte and endothelial celladhesion proteins and cell specific chemoattractants and activatingfactors (Springer, T. A., Cell, 76:301-314 (1994); Butcher, E. C., Cell,67:1033-1036 (1991); Butcher, E. C. and Picker, L. J., Science (Wash.DC), 272:60-66 (1996)). These are: (a) a low affinity interactionbetween leukocyte selectins and endothelial cell carbohydrates; (b) ahigh-affinity interaction between leukocyte chemoattractant receptorsand chemoattractant/activating factors; and (c) a tight-binding betweenleukocyte integrins and endothelial cell adhesion proteins of theimmunoglobulin superfamily. Different leukocyte subsets expressdifferent repertoires of selectins, chemoattractant receptors andintegrins. Additionally, inflammation alters the expression ofendothelial adhesion proteins and the expression of chemoattractant andleukocyte activating factors. As a consequence, there is a great deal ofdiversity for regulating the selectivity of leukocyte recruitment toextravascular sites. The second step is crucial in that the activationof the leukocyte chemoattractant receptors is thought to cause thetransition from the selectin-mediated cell rolling to theintegrin-mediated tight binding. This results in the leukocyte beingready to transmigrate to perivascular sites. Thechemoattractant/chemoattractant receptor interaction is also crucial fortransendothelial migration and localization within a tissue (Campbell,J. J., et al., J. Cell Biol., 134:255-266 (1996); Carr, M. W., et al.,Immunity, 4:179-187 (1996)). This migration is directed by aconcentration gradient of chemoattractant leading towards theinflammatory focus.

[0185] CCR2 has an important role in leukocyte trafficking. It is likelythat CCR2 is a key chemokine receptor for T cell or T cell subset ormonocyte migration to certain inflammatory sites, and so anti-CCR2 mAbscan be used to inhibit (reduce or prevent) T cell or monocyte migration,particularly that associated with T cell dysfunction, such as autoimmunedisease, or allergic reactions or with monocyte-mediated disorders suchas atherosclerosis. Accordingly, the antibodies and fragments thereof ofthe present invention can also be used to modulate receptor function inresearch and therapeutic applications. For instance, the antibodies andfunctional fragments described herein can act as inhibitors to inhibit(reduce or prevent) (a) binding (e.g., of a ligand, an inhibitor or apromoter) to the receptor, (b) a receptor signaling function, and/or (c)a stimulatory function. Antibodies which act as inhibitors of receptorfunction can block ligand or promoter binding directly or indirectly(e.g., by causing a conformational change). For example, antibodies caninhibit receptor function by inhibiting binding of a ligand, or bydesensitization (with or without inhibition of binding of a ligand).Antibodies which bind receptor can also act as agonists of receptorfunction, triggering or stimulating a receptor function, such as asignaling and/or a stimulatory function of a receptor (e.g., leukocytetrafficking) upon binding to receptor.

[0186] Thus, the present invention provides a method of inhibitingleukocyte trafficking in a mammal (e.g., a human patient), comprisingadministering to the mammal an effective amount of an antibody orfunctional fragment of the present invention. Administration of anantibody or fragment of the present invention can result in ameliorationor elimination of the disease state.

[0187] The antibody of the present invention, or a functional fragmentthereof, can also be used to treat disorders in which activation of theCCR2 receptor by binding of chemokines is implicated. For example, theantibodies or functional fragments thereof (e.g., 1D9 and/or 8G2 orfunctional fragments thereof) can be used to treat allergy,atherogenesis, anaphylaxis, malignancy, chronic and acute inflammation,histamine and IgE-mediated allergic reactions, shock, and rheumatoidarthritis, atherosclerosis, multiple sclerosis, stenosis, restenosis,allograft rejection, fibrotic disease, asthma, and inflammatoryglomerulopathies.

[0188] Diseases or conditions of humans or other species which can betreated with inhibitors of CCR2 receptor function (including antibodiesor suitable fragments thereof), include, but are not limited to:

[0189] inflammatory or allergic diseases and conditions, includingrespiratory allergic diseases such as asthma, allergic rhinitis,hypersensitivity lung diseases, hypersensitivity pneumonitis,interstitial lung diseases (ILD) (e.g., idiopathic pulmonary fibrosis,or ILD associated with rheumatoid arthritis, systemic lupuserythematosus, ankylosing spondylitis, systemic sclerosis, Sjogren'ssyndrome, polymyositis or dermatomyositis); anaphylaxis orhypersensitivity responses, drug allergies (e.g., to penicillin,cephalosporins), insect sting allergies; inflammatory bowel diseases,such as Crohn's disease and ulcerative colitis; spondyloarthropathies;scleroderma; psoriasis and inflammatory dermatoses such as dermatitis,eczema, atopic dermatitis, allergic contact dermatitis, urticaria;vasculitis (e.g., necrotizing, cutaneous, and hypersensitivityvasculitis);

[0190] autoimmune diseases, such as arthritis (e.g., rheumatoidarthritis, psoriatic arthritis), multiple sclerosis, systemic lupuserythematosus, myasthenia gravis, juvenile onset diabetes, nephritidessuch as glomerulonephritis, autoimmune thyroiditis, Behcet's disease;

[0191] graft rejection (e.g., in transplantation), including allograftrejection or graft-versus-host disease, and organ transplant-associatedarteriosclerosis;

[0192] atherosclerosis;

[0193] cancers with leukocyte infiltration of the skin or organs;

[0194] stenosis or restenosis of the vasculature, particularly of thearteries, e.g., the coronary artery, such as stenosis or restenosiswhich results from vascular intervention (e.g., surgical, therapeutic ormechanical intervention), as well as neointimal hyperplasia. Forexample, restenosis, which typically produces a narrowing of the lumenalopening of the vessel, can result from vascular injury including, butnot limited to, that produced by vascular graft procedures, angioplasty,including angioplasty performed by balloon, atherectomy, laser or othersuitable method (e.g., percutaneous translumenal coronary angioplasty(PTCA)), stent placement (e.g., mechanical or biological endovascularstent placement), vascular bypass procedures or combinations thereof, aswell as other procedures used to treat stenotic or occluded bloodvessels;

[0195] other diseases or conditions (including CCR2-mediated diseases orconditions), in which undesirable inflammatory responses are to beinhibited can be treated, including, but not limited to, reperfusioninjury, certain hematologic malignancies, cytokine-induced toxicity(e.g., septic shock, endotoxic shock), polymyositis, dermatomyositis,and granulomatous diseases including sarcoidosis.

[0196] Diseases or conditions of humans or other species which can betreated with promoters of CCR2 receptor function (including antibodiesor fragments thereof), include, but are not limited to:

[0197] immunosuppression, such as that in individuals withimmunodeficiency syndromes such as A1DS, individuals undergoingradiation therapy, chemotherapy, therapy for autoimmune disease or otherdrug therapy (e.g., corticosteroid therapy), which causesimmunosuppression; and immunosuppression due congenital deficiency inreceptor function or other causes.

[0198] Anti-CCR2 antibodies of the present invention can block thebinding of one or more chemokines, thereby blocking the downstreamcascade of one or more events leading to the above disorders.

[0199] Antibodies and functional fragments thereof which are antagonistsof CCR2 can be used as therapeutics for A1DS, as well as certaininflammatory diseases. HIV-1 and HIV-2 are the etiologic agents ofacquired immunodeficiency syndrome (A1DS) in humans. A1DS results inpart from the depletion of CD4+ T lymphocytes in HIV infectedindividuals. HIV-1 infects primarily T lymphocytes,monocytes/macrophages, dendritic cells and, in the central nervoussystem, microglia. All of these cells express the CD4 glycoprotein,which serves as a receptor for HIV-1 and HIV-2. Efficient entry of HIVinto target cells is dependent upon binding of the viral exteriorenvelope glycoprotein, gp120, to the amino-terminal CD4 domain. Aftervirus binding, the HIV-1 envelope glycoproteins mediate the fusion ofviral and host cell membranes to complete the entry process. Membranefusion directed by HIV-1 envelope glycoproteins expressed on theinfected cell surface leads to cell-cell fusion, resulting in syncytia.

[0200] Recently, host cell factors in addition to CD4 have beensuggested to determine the efficiency of HIV-1 envelopeglycoprotein-mediated membrane fusion. The 7 transmembrane receptor(7TMR) termed HUMSTSR, LESTR, or “fusin” has been shown to allow a rangeof CD4-expressing cells to support infection and cell fusion mediated bylaboratory-adapted HIV-1 envelope glycoproteins (Feng, Y., et al.,Science (Wash. DC), 272:872-877 (1996)). Antibodies to HUMSTSR blockedcell fusion and infection by laboratory-adapted HIV-1 isolates but notby macrophage-tropic primary viruses in vitro (Feng, Y., et al., Science(Wash. DC), 272:872-877 (1996)).

[0201] The ability of chemokine receptors and related molecules tofacilitate the infection of primary clinical HIV-1 isolates has beenreported recently by several groups (see e.g., Bates, P., Cell, 86:1-3(1996); Choe, H., et al., Cell, 85:1135-1148 (1996); Doranz et al., Cell85:1149-1158 (1996)). These studies indicated that involvement ofvarious members of the chemokine receptor family in the early stages ofHIV-1 infection helps to explain viral tropism and β-chemokineinhibition of primary HIV-1 isolates.

[0202] The present invention also provides a method of inhibiting HIVinfection of a cell (e.g., new infection and/or syncytium formation)which expresses a mammalian CCR2 or portion thereof, comprisingcontacting the cell with a composition comprising an effective amount ofan antibody or functional fragment thereof which binds to a mammalianCCR2 or portion of said receptor. The composition can also comprise oneor more additional agents effective against HIV, including, but notlimited to, anti-CCR3 antibodies, anti-CCR5 antibodies, and anti-fusinantibodies.

[0203] Various methods can be used to assess binding of HIV to a celland/or infection of a cell by HIV in the presence of the antibodies ofthe present invention. For example, assays which assess binding of gp120or a portion thereof to the receptor, HIV infection and syncytiumformation can be used (see, for example, Choe, H., et al., Cell,85:1135-1148 (1996)). The ability of the antibody of the presentinvention to inhibit these processes can be assessed using these orother suitable methods.

[0204] In addition, the present invention provides a method of treatingHIV in a patient, comprising administering to the patient a compositioncomprising an effective amount of an antibody or functional fragmentthereof which binds to a mammalian CCR2 or portion of said receptor.Again, the composition can also comprise one or more additional agentseffective against HIV, including, but not limited to, anti-CCR3antibodies, anti-CCR5 antibodies, and anti-fusin antibodies. Therapeuticuse of antibody to treat HIV includes prophylactic use (e.g., fortreatment of a patient who may be or who may have been exposed to HIV).For example, health care providers who may be exposed or who have beenexposed to HIV (e.g., by needle-stick) can be treated according to themethod. Another example is the treatment of a patient exposed to virusafter unprotected sexual contact or failure of protection.

[0205] In AIDS, multiple drug treatment appears the most promising. Ananti-chemokine receptor antagonist that inhibits HIV infection can beadded to the drug treatment regimen, in particular by blocking virusinfection of new cells. Thus, administration of an antibody or fragmentof the present invention in combination with one or more othertherapeutic agents such as nucleoside analogues (e.g., AZT, 3TC, ddI)and/or protease inhibitors is envisioned, and provides an importantaddition to an HIV treatment regimen. In one embodiment, a humanizedanti-CCR2 mAb is used in combination with a (i.e., one or more)therapeutic agent to reduce viral load from patients, by preventingfusion and/or infection of new cells. Such an antibody can also beuseful in preventing perinatal infection.

[0206] Another aspect of the invention relates to a method of preventingHIV infection in an individual, comprising administering to theindividual an effective amount of an antibody or functional fragmentthereof which binds to CCR2. According to the method, preventing HIVinfection includes treatment in order to prevent (reduce or eliminate)infection of new cells in an infected individual or in order to preventinfection in an individual who may be, may have been, or has been,exposed to HIV. For example, individuals such as an HIV infectedindividual, a fetus of an HIV infected female, or a health care workermay be treated according to the method of the present invention.

[0207] Modes of Administration

[0208] One or more antibodies or fragments of the present invention canbe administered to an individual by an appropriate route, either aloneor in combination with (before, simultaneous with, or after) anotherdrug or agent, or before, simultaneous with or after surgical,mechanical or therapeutic intervention. For example, the antibodies ofthe present invention can also be used in combination with othermonoclonal or polyclonal antibodies (e.g., in combination withantibodies which bind other chemokine receptors, including, but notlimited to, CCR3 and CCR5) or with existing blood plasma products, suchas commercially available gamma globulin and immune globulin productsused in prophylactic or therapeutic treatments. The antibodies orfragments of the present invention can be used as separatelyadministered compositions given in conjunction with antibiotics and/orantimicrobial agents.

[0209] An effective amount of an antibody or fragment (i.e., one or moreantibodies or fragments) is administered. An effective amount is anamount sufficient to achieve the desired therapeutic (includingprophylactic) effect, under the conditions of administration, such as anamount sufficient for inhibition of a CCR2 function, and thereby,inhibition of an inflammatory response or HIV infection, or an amountsufficient for promotion of a CCR2 function, as indicated.

[0210] A variety of routes of administration are possible including, butnot necessarily limited to, oral, dietary, topical, parenteral (e.g.,intravenous, intraarterial, intramuscular, subcutaneous injection orinfusion), inhalation (e.g., intrabronchial, intraocular, intranasal ororal inhalation, intranasal drops), depending on the disease orcondition to be treated. Other suitable methods of administration canalso include rechargeable or biodegradable devices and slow releasepolymeric devices. The pharmaceutical compositions of this invention canalso be administered as part of a combinatorial therapy with otheragents.

[0211] Formulation of an antibody or fragment to be administered willvary according to the route of administration and formulation (e.g.,solution, emulsion, capsule) selected. An appropriate pharmaceuticalcomposition comprising an antibody or functional fragment thereof to beadministered can be prepared in a physiologically acceptable vehicle orcarrier. A mixture of antibodies and/or fragments can also be used. Forsolutions or emulsions, suitable carriers include, for example, aqueousor alcoholic/aqueous solutions, emulsions or suspensions, includingsaline and buffered media. Parenteral vehicles can include sodiumchloride solution, Ringer's dextrose, dextrose and sodium chloride,lactated Ringer's or fixed oils. A variety of appropriate aqueouscarriers are known to the skilled artisan, including water, bufferedwater, buffered saline, polyols (e.g., glycerol, propylene glycol,liquid polyethylene glycol), dextrose solution and glycine. Intravenousvehicles can include various additives, preservatives, or fluid,nutrient or electrolyte replenishers (See, generally, Remington'sPharmaceutical Science, 16th Edition, Mack, Ed. 1980). The compositionscan optionally contain pharmaceutically acceptable auxiliary substancesas required to approximate physiological conditions such as pH adjustingand buffering agents and toxicity adjusting agents, for example, sodiumacetate, sodium chloride, potassium chloride, calcium chloride andsodium lactate. The antibodies and fragments of this invention can belyophilized for storage and reconstituted in a suitable carrier prior touse according to art-known lyophilization and reconstitution techniques.The optimum concentration of the active ingredient(s) in the chosenmedium can be determined empirically, according to procedures well knownto the skilled artisan, and will depend on the ultimate pharmaceuticalformulation desired. For inhalation, the antibody or fragment can besolubilized and loaded into a suitable dispenser for administration(e.g., an atomizer, nebulizer or pressurized aerosol dispenser).

[0212] The present invention will now be illustrated by the followingExamples, which are not intended to be limiting in any way. Theteachings of all references cited herein are incorporated herein byreference.

EXAMPLES Example 1

[0213] Materials:

[0214] The following materials were obtained from the indicated sources:

[0215] PE-conjugated anti-CD16, PE-conjugated streptavidin, andbiotinylated anti-human IgE were from Pharmingen (San Diego, Calif.).FITC-conjugated goat anti-mouse IgG was from Jackson ImmunoresearchLaboratories (West Grove, Pa.). FACS Lysing Buffer was from BectonDickenson (Mountain View, Calif.) and [¹²⁵I]-MCP-1 was from NEN (Boston,Mass.).

[0216] Cells, Cell Lines, and Tissue Culture

[0217] The murine pre-B lymphoma cell line L1/2 was maintained inRPMI-1640 supplemented with 10% Fetal Clone I (Gibco BRL, Gaithersburg,Md.) 50 Units/mL penicillin (Gibco BRL), 50 μg/mL streptomycin (GibcoBRL), 2 mM L-Glutamine (Gibco BRL), and 55 μM β-mercaptoethanol (GibcoBRL). Other cell lines included transfectants of L1/2 cells expressingeither CCR1 (Campbell, J. et al. (1996) J. Cell Bio., 134:255-266), CCR5(Wu et al., Nature 384:179-183 (1996)) grown in the above culture mediumsupplemented with 800 μg/ml active G418. THP-1 cells (ATCC No. TIB202)were grown in accordance with ATCC instructions. PBMC were purified fromheparinized blood as described in Ponath et al., J. Clin. Invest.,97:604-612 (1996).

[0218] Preparation of CCR2b Expression Construct and StableTransfectants

[0219] The coding region for the human CCR2b (Charo et al. (1994) Proc.Natl. Acad. Sci. USA, 91:2752) was obtained by RT-PCR amplification asdescribed (Qin, S. et al. (1996) Eur. J. Immunol., 26:640-647). cDNA wasmade using oligo(dT)-priming, and amplification of the CCR2b codingregion was achieved by nested PCR with the following sets of primerswhich correspond to the positions of the CCR2b sequence (GenBankAccession No. U03905; Charo et al., Proc. Natl. Acad. Sci. USA91:2752-2756 (1994)) as indicated: 1) 5′ primer:5′-TGAGACAAGCCACAAGCTGAAC-3′ (nucleotides 11 to 32; SEQ ID NO: 1); 3′Primer: 5′-TCTGTATTAGTACACACAGCCC-3′ (nucleotides 1301 to 1280; SEQ IDNO: 2); 2) 5′ Primer: 5′-ATGCTGTCCACATCTCGTTCTCGG-3′ (nucleotides 81 to104; SEQ ID NO: 3); 3′ Primer: 5′-TTATAAACCAGCCGAGACTTCCTGCTC-3′(nucleotides 1164 to 1137; SEQ ID NO: 4).

[0220] The CCR2B cDNA coding region was modified to contain the CD5signal peptide leader sequence (Aruffo et al., Cell 61:1303-1313(1990)). The predicted amino acid sequence of this peptide is:

[0221]NH₂-Met-Pro-Met-Gly-Ser-Leu-Gln-Pro-Leu-Ala-Thr-Leu-Tyr-Leu-Leu-Gly-Met-Leu-Val-Ala-Ser-Val-Leu-Ala. . . (SEQ ID NO: 5)

[0222] Using PCR with the CCR2b cDNA as template and two overlapping 5′primers that contain a BamHI restriction site, encode the CD5 signalpeptide sequence and the amino terminal sequence of CCR2b, and a 3′primer located internally in the CCR2b coding region. 5′CD5 Seq1 primer5′-GGGGATCCAGAAACCATGCCCATGGGGTCT CTGCAACCGCTGGCCACCTTGTACCTGCTG-3′ (SEQID NO: 6) 5′CD5 Seq2 primer 5′-GCCACCTTGTACCTGCTGGGGATGCTGGTCGCTTCCGTGCTAGCGATGCTGTCCACATCTCGTTC-3′ (SEQ ID NO: 7) 3′CCR2AB2 primer -5′-GACGACCAGCATGTTGCC-3′ (SEQ ID NO: 8; U03905 nucleotides 272 to 255)

[0223] The 278 base pair amplified fragment was digested with BamHI andApal and the resulting 209 base pair fragment was inserted at the Apalsite at position 206 of the CCR2b cDNA (GenBank Accession No. U03905) toreplace the endogenous 5′ base pair fragment of CCR2. The resultingsequence that encodes a CCR2b with the CD5 signal peptide leadersequence immediately preceding the receptor initiator methionine wasinserted into the BamHI and Xhol sites of pcDNA3 (Invitrogen, San Diego,Calif.) to create the mammalian expression plasmid pCD5MCPRB. TheCD5-CCR2b fragment was subcloned into the BamH I-Not I site of pCDEF3(Goldman et al., (1 996) Biotechniques 21:1013-1015), and this constructwas designated CCR2bDEF3. In this expression vector, the expression ofthe inserted gene is driven by the EF-1α promoter.

[0224] Fifty milliliters of L1/2 cells were seeded at 4×10⁵ cells/mL theday before the electroporation. On the day of the electroporation, thecells, which had grown up to a density of 1×10⁶/mL, were centrifuged outof their medium and resuspended in 800 μl room temperatureelectroporation buffer (Zajac et al., DNA 7:509-513). 120 mM L-GlutamicAcid (Sigma), 7 mM Mg Acetate (EM Science), 4.3 mM Glucose (Sigma), 17mM K Pipes, pH 6.9 (Sigma), 1 mM EGTA (Sigma), 5 mM ATP, pH 7.0 (Sigma).Twenty-five micrograms Sca I linearized, phenol/chloroform/isoamylalcohol extracted and isopropanol precipitated CCR2bDEF3 plasmid DNA wasplaced in an 0.4 cm gap electroporation curvette. The resuspended cellswere added to the curvette, and a single pulse applied at 450 volts, 960μFd. The cells were then transferred from the curvette to a T-75 flaskcontaining 15 mL L1/2 growth medium (described above, and grown forthree days, at which time the cells were centrifuged out of their mediumand resuspended in L1/2 growth medium additionally supplemented with 1mM sodium pyruvate (Gibco BRL) and 0.8 mg/mL active G418 (Gibco BRL).

[0225] Selection of Cells Expressing CCR2b by Chemotaxis

[0226] The transfected cells were allowed to grow for eleven days, atwhich point they were split 1:20 into fresh growth medium. On thesixteenth day, the cells were selected by chemotaxis. 600 μL 1 nM MCP-1in RPMI 1640 supplemented with 0.5% BSA (RPMI/BSA) was placed in thelower chamber and 1×10⁶ CCR2bDEF3 cells in 100 μl of RPMI/BSA wereplaced in the upper chamber of a 3.0 micron pore 24-well chemotaxisplate (Becton Dickinson). The cells were allowed to chemotax for fourhours and twenty minutes in a 37° C, 5% CO₂, humidified incubator, atwhich time the upper chamber was removed. This incubation time waschosen at the time of the experiment because it was sufficiently longfor cells responding to the MCP-1 to chemotax, but short enough to keepthe background low.

[0227] Secondary Selection of CCR2b Expressing-Cells by FACS Sorting

[0228] The cells which had chemotaxed through the membrane and into thelower chamber were grown up, and further purified by sterile FACSsorting. Ten million CCR2bDEF3 cells were centrifuged out of theirmedium, resuspended in 2.5 mL PBS(+Ca, Mg) supplemented with 1%heat-inactivated Fetal Calf Serum (“HI FCS”) (Gibco BRL) and 2.5 mLsterile filtered anti-CCR2b amino-terminal peptide antibody supernatant5A11. The cells and the antibody were mixed and allowed to incubate onice for thirty minutes. The cells were then washed twice with PBS (+)(Gibco BRL), and resuspended in 5 mL of a sterile filtered, 1:250dilution of FITC-conjugated, affinity-purified F(ab¹)₂ goat anti-mouselgG (Jackson ImmunoResearch Laboratories) in PBS (+) supplemented with1% HI FCS. The cells were incubated for thirty minutes on ice in thedark, and then washed twice with PBS(+) (GIBCO BRL). The cells weresorted on the FACSCalibur® and the brightest 4% of cells were collected.(FL1≧3×10²).

[0229] The sorted cells were allowed to grow up, and they were resortedusing the same protocol as above. The brightest 1% of cells werecollected. (FL1≧3×10³).

[0230] Monoclonal Antibody Production

[0231] To produce mAbs to CCR2b, transfectants were continuallymonitored to ensure that levels of expression did not drift downward.FACS staining was performed periodically to ascertain receptorexpression on the transfectants using the anti CCR2b antibodysupernatant 5A11 with goat anti-mouse IgG FITC as the secondaryantibody.

[0232] Twenty million CCR2bDEF3.L1/2 cells were washed in RPMI 1640(Gibco BRL) and incubated in RPMI 1640 plus 0.2 mg/mL Mitomycin C for 30minutes at 37° C. The cells were then washed twice with PBS (+) and2×10⁷ cells in 0.5 mL PBS (+) were injected intraperitoneally into a C57BL/6 female mouse. This was repeated two more times at two weekintervals. The fourth time, 2×10⁷ cells were resuspended in 0.25 mL andinjected intravenously. Three days after the intravenous injection, themouse was sacrificed and the spleen removed and the cells fused with theSP2/0 cell line as described (Current Protocols in Immunology, JohnWiley and Sons, New York, 1992).

[0233] This set of mice had previously been immunized many times with 2different cell lines as well as a synthetic peptide, but no antibodiesthat stained CCR2 positive cells were generated from several fusions.The above four immunizations with the CCR2bDEF3.L1/2 cell lineexpressing high levels of CCR2b were critical to obtain the describedantibody.

[0234] Selecting Single Cell Clone of CCR2 Transfectants by LimitingDilution

[0235] After the mouse received the last injection, the twice sortedcells were allowed to grow up again, and then they were further purifiedby limiting dilution. The cells were plated at 1 and 0.5 cell per wellin 96 well plates. Subcloned cells from the 0.5 cell per well dilutionwere grown up and tested for CCR2b expression by indirectimmunofluorescent FACS analysis using the anti-CCR2b antibodysupernatant 5A11 with goat anti-mouse IgG FITC as the secondaryantibody. The procedure was the same as described above, except that thestaining volume was 100 μl. Four positives were selected and frozendown.

[0236] Identification of Positive Monoclonal Antibodies

[0237] Immunofluorescent staining analysis using a FACScan® (BectonDickinson & Co., Mountain View, Calif.) was used to identify themonoclonal antibodies which were reactive with the CCR2b receptor.Hybridoma culture supernatants were assayed in a 96-well format usinggoat anti-mouse IgG FITC as the secondary antibody. CCR2bDEF3.L1/2 cellswere used to identify monoclonal antibodies reactive with CCR2b, anduntransfected L1/2 cells were used to eliminate monoclonal antibodiesreactive with other cell surface proteins.

[0238] FACS Staining—Cultured Cells

[0239] For the staining of cultured transfectant cell lines 0.5×10⁶cells in 50 μl were resuspended in PBS+1% FCS in a 96 well polystyreneV-bottom plate. 50 μl of primary antibody supernatants or HT medium(negative control) were added, and the samples were incubated at 4° C.for 30 min. 100 μl of PBS were added and the cells were pelleted bycentrifugation and washed once with PBS. The pellet was resuspended in100 μl PBS+1% FCS containing FITC-conjugated goat anti-mouse IgGantibody (a 1:250 dilution) and incubated for thirty minutes at 4° C. inthe dark. The cells were washed twice with PBS, resuspended in PBS, andanalyzed by flow cytrometry with a FacScan cytometer using the CellQuestsoftware (Becton-Dickenson) Cells were fixed with PBS/1% formaldehyde ifthey were not to be analyzed the same day. Monoclonal antibodies 1D9 and8G2 stain CCR2 transfectants but not CCR1 or CCR5 transfectants (FIG.1).

[0240] FACS Staining—Whole Blood

[0241] 100 μl whole blood was mixed with 100 μL of 1D9 antibodyhybridoma supernatants or HT medium (negative control) and incubated at4° C. for 30 min. After one wash with PBS, 100 μL FITC-conjugated goatanti-mouse IgG antibody (a 1:250 dilution) was added to each sample andincubated for 30 min. at 4° C. in the dark. Samples were then washedonce with PBS if a second color staining is to be done, otherwise washedtwice more in PBS. For two color staining 5 μl of mouse serum was addedto the cell pellets after the single wash, mixed, and incubated for fiveminutes at 4° C. in the dark. Second primary antibodies (or PBS as anegative control) were added (10 μl anti-CD 16, 100 μl 1:200 dilution ofanti-IgE) and incubated for thirty minutes at 4° C. in the dark. Sampleswere then washed one time with PBS and resuspended in 100 μLstreptavidin PE (1:200 PBS+1% BSA) and incubated for fifteen minutes at4° C. in the dark. Eyrythrocytes were lysed by adding 2 ml of FACSLysing Buffer to each sample and incubating at room temperature in thedark for fifteen minutes or until samples were clear. The cells werepelleted by centrifugation and all but 200 μl of the supernatant wasaspirated. The samples were analyzed by flow cytometry on a FacScancytometer using the CellQuest software (Becton-Dickenson). CCR2b isexpressed on most monocytes, a subpopulation of lymphocytes and a subsetof granulocytes (FIGS. 2A-2L). CCR2b is expressed on an IgE-positivepopulation in peripheral blood (basophils)(FIGS. 3A-3I).

[0242] MCP-1 Binding Assays

[0243] MCP-1 binding was performed in a final volume of 0.1 ml of 50 mMHepes pH 7.4, 1 mM CaCl₂, 5 mM MgCl₂, 0.02% sodium azide, 0.5% BSA(HBB), containing either 2.5 μg THP-1 membrane protein or 500,000 PBMCand 0.1 nM of [¹²⁵I]-MCP-1. Competition binding experiments wereperformed by including variable concentrations of unlabeled MCP-1, 1D9antibody, or a negative control IgG2a. Nonspecific binding wasdetermined following the addition of a 2500-fold excess of unlabeledMCP-1. Samples were incubated for 60 minutes at room temperature, andbound and free tracer were separated by filtration through 96-well GF/Bfilterplates presoaked in 0.3% polyethyleneimine. The filters werewashed in HBB further supplemented with 0.5 M NaCl, dried, and theamount of bound radioactivity determined by liquid scintillationcounting. mAb 1D9 inhibits [¹²⁵I ]MCP-1 binding to THP-1 cell membraneswith an IC₅₀ of about 0.004 μg/ml (approximately 0.02 nM; FIG. 4) and tofresh PBMC with an lC₅₀ of 0.04 μg/ml (approximately 0.2 nM; FIG. 5).

[0244] Chemotaxis of PBMC

[0245] Chemotaxis was assayed using a 3 μm pore size 96-well chemotaxisplate (Neuroprobe, Cabin John, Md.). PBMC isolated by standard methodsusing Ficoll-Hypaque density gradient centrifugation were washed withPBS/0.5%BSA and then resuspended in chemotaxis assay media (HBSS/10 mMHEPES/0.5% Fatty acid free BSA) to a final concentration of 10×10⁶cells/ml. Cells were princubated in chemotaxis assay media at roomtemperature for 20 min. with various concentrations of the anti-CCR2antibody, 1D9, or nonspecific murine IgG2a. The same dilutions ofantibody were mixed with chemokine and 30 μl of the mixture was added toeach of the bottom wells of the chemotaxis plate. The bottom wells arecovered with the membrane, and 25 μl of the cell and antibody mixtureare added to the top of the filter. The plates are incubated at 37° C.in 5% CO₂ incubator for approximately 80 min. At the completion of themigration, the membrane is removed and the plate with the bottom wellsis incubated −80 C for 30 minutes to freeze the contents. The plates arethawed at 37° C. for 10 minutes. 6 μl of a 1:400 dilution of CyQuantreagent (Molecular Probes, Eugene, Oreg.) in a lysis buffer provided bythe supplier is added to each well, and the cell migration is quantifiedas indicated by fluorescence intensity determined using a CytoFlourfluorescence plate reader at 485ex/535em. mAb 1D9 inhibits MCP-1-inducedchemotaxis, but not RANTES-induced chemotaxis, of fresh PBMC (FIGS. 6Aand 6B). Inhibition of MCP-1-induced chemotaxis of fresh PBMC has beendemonstrated with 10 μg/ml (≈40 nM).

Example 2

[0246] Humanisation of Monoclonal Antibody 1D9

[0247] The 1D9 monoclonal antibody is likely to be immunogenic inhumans, potentially eliciting a human anti-mouse antibody (HAMA)response. This HAMA response usually results in rapid clearance of themouse monoclonal antibody from the body, thus limiting any therapeuticeffect the 1D9 monoclonal antibody might have. Therefore, in an effortto reduce the immunogenicity of this antibody in humans and to maximizeits therapeutic potential, the humanisation of the 1D9 mouse monoclonalantibody was undertaken. The following examples provide a detailedanalysis of the 1D9 amino acid sequence data, the building of amolecular model of the murine 1D9 F_(V) domain, and the design strategyfor the successful humanization of the mouse antibody. This designstrategy resulted in the design of a number of humanized versions ofboth the kappa light chain variable (V_(K)) region and the heavy chainvariable (V_(H)) region. In total, the humanized V_(H) region includedup to 16 amino acid changes in the FRs of the selected human V_(H)region. These changes were subdivided between four versions of thehumanized V_(H) region. In addition, twelve amino acid changes in theFRs of the selected human V_(K) region were included in the fourversions of the humanized VK region which were also designed.

[0248] Sequence Analysis of the Mouse 1D9 Kappa Light Chain VariableRegion

[0249] The amino acid sequence of the 1D9 V_(K) region (FIG. 7) wascompared with other mouse kappa light chain variable regions and alsothe consensus sequences of the subgroups that the variable regions weresubdivided into in the Kabat database (Kabat et al., Sequences ofproteins of immunological interest, Fifth edition, U.S. Department ofHealth and Human Services, U.S. Government Printing Office (1991)). Fromthis analysis the 1D9 V_(K) region was found to most closely match themouse consensus sequence of mouse kappa subgroup II (Identity=79.46%,Similarity=82.14%). When only the FRs of the 1D9 kappa light chainvariable region were compared in the mouse subgroup II, percentageidentity increased to 87.5%, while percentage similarity increased to88.75%. In addition, the mouse 1D9 V_(K) region showed good homology toa translation of the 70/3 murine V_(K) germline gene (FIG. 13). Takentogether, the above evidence clearly proved that the 1D9 sequence wastypical of a mouse V_(K) region.

[0250] Sequence Analysis of the Mouse 1D9 Heavy Chain Variable Region

[0251] A similar analysis of the 1D9 V_(H) region (FIG. 8) found that itmatched closest to the consensus sequence of the mouse heavy chainsubgroup IIIc in the Kabat database (Kabat et al., Sequences of proteinsof immunological interest, Fifth edition, U.S. Department of Health andHuman Services, U.S. Government Printing Office (1991)). Identitybetween the mouse heavy chain variable region amino acid sequence of 1D9and the consensus sequence of mouse subgroup IIIc was measured at70.94%, while the similarity was calculated to be 76.07%. When only theFRs of the 1D9 V_(H) region was compared to mouse subgroup IIIc,percentage identity increased to 75.86%, while the similarity increasedto 80.46%. The mouse 1D9 V_(H) region also showed good homology to atranslation of the MLR-RF24BG murine V_(H) germline gene, among others(FIG. 14). Thus, the above evidence confirmed that the 1D9 sequence wastypical of a mouse V_(H) region.

[0252] Molecular Modelling of the 1D9 Domain.

[0253] To assist in the design of the humanized variable regions of the1D9 antibody, a series of molecular models were constructed of themurine 1D9 F_(V) region and the eight CDR grafted variants. This wasdone using the AbM molecular modeling package supplied and utilized byOxford Molecular Limited (OML). Antibody x-ray crystallographicstructures available from the Brookhaven database were formatted toallow them to be used for modeling with AbM.

[0254] The FRs of the 1D9 variable regions were modeled on FRs fromsimilar, structurally-solved immunoglobulin variable regions. Whileidentical amino acid side chains were kept in their originalorientation, mismatched side chains were substituted as in the original1D9 F_(V). The backbone atoms of the FRs of the Fab BvO4-01 V_(K) regionwere used for the model of the Fv framework region of 1D9 for both theV_(K) and V_(H) chains (Brookhaven PDB code 1nbv, solved to 2.0 Å). Thesequence of Fab Bv04-01 was a good match for the variable regionsequences of murine 1D9 and their humanized variants. The identitiesbetween Fab Bv04-01 and the murine 1D9 and humanized sequences rangedfrom 76% to 78% for V_(K) sequences and from 74% to 84% for V_(H)sequences. Testing of AbM with known structures has shown that FRbackbone homology is an important factor in the quality of any model,since the use of FR structures that poorly match a sequence beingmodeled can significantly and adversely affect the position andorientation of the CDR loops.

[0255] For the backbone structures of CDRs L1, L2, L3, H1 and H2,conformations for all of the models were taken from canonical classesused by AbM without modification, using the classes shown in FIGS. 9 and10.

[0256] For the backbone structure of the L1 loop, the loop conformationsof the murine 1D9 VK region was taken from canonical Class 4 from AbM.This canonical class is based on those described by Chothia and hiscolleagues (Chothia and Lesk, J. Mol. Biol. 197:901 (1987); Chothia etal., Nature 34:877 (1989); Tramontano et al., i J. Mol. Biol. 215:175(1990); and Chothia et al., J. Mol. Biol. 227:799 (1992)), but they havebeen modified to take into consideration structures that have becomeavailable since the original articles were published. Testing of theperformance of AbM predictions for known loop structures has shown thatCDR loops which are created in this way are usually modeled veryaccurately, i.e. to within 1-1.5 Å RMS deviation.

[0257] The H3 loop in the 1D9 V_(H) region is comparatively short at sixresidues long. It was modeled using a search for backbone conformationsfrom X-ray structures in the Brookhaven databank. For short loops likethis, there are sufficient loop conformations from known X-raystructures to saturate the conformational space available to the loop.Testing of the predictions made by AbM with the structures of newantibodies, where the structure is not included in the databases used bythe program, shows that for CDR H3 loops of this size, the accuracy islikely to be at least 2.0 Å.

[0258] After adjusting the whole of the model for obvious steric clashesit was subjected to energy minimisation, as implemented in MACROMODEL,both to relieve unfavorable atomic contacts and to optimize van derWaals and electrostatic interactions.

[0259] Design of the Humanised 1D9 V_(K) Antibody Variants.

[0260] The first step in the design of the humanised variable regions ofthe 1D9 antibody was the selection of the human kappa light chainvariable region that would serve as the basis for the humanized 1D9V_(K), region. As an aid to this process the 1D9 V_(K) region wasinitially compared to the consensus sequence of the four human kappalight chain variable region subgroups as defined by Kabat (Kabat et al.,Sequences of proteins of immunological interest, Fifth edition, U.S.Department of Health and Human Services, U.S. Government Printing Office(1991)). The mouse 1D9 light chain variable region was most similar tothe consensus sequence of human kappa light chain subgroup II, withwhich it displayed a 76.2% identity over the whole variable region and a82.5% identity within the FRs alone. When measured with respect tosimilarity, these values increased to 79.7% overall and 85.0% within theFRs alone. Consequently it generally appeared to match well to humankappa light chain variable region sequences from kappa subgroup II.

[0261] The mouse 1D9 V_(K) was then compared to all the recordedexamples of individual sequences of human variable regions publiclyavailable. FIG. 15 shows the best seventeen matches to the mouse 1D9V_(K) region which were identified through this analysis. Overall, thesearch algorithm selected the human V_(K) region antibody 036521(Rheinnecker et al., Journal of Immunology. 157(7):2989-97 (1996)) asthe best match to the mouse 1D9 V_(K) region (FIG. 16). However, areview of the source paper for this antibody revealed that murineoligonucleotide primers had been used to rescue the genes from thehybridoma. This meant that this antibody was in fact a murine antibodyand not human, as suggested by the Kabat database. Thus,the next bestmatch to the murine 1D9 V_(K) region that was selected by the databasesearch was the human V_(K) region from antibody HF-21/28 (Kabat database1D number 005056; Chastagner et al., Gene. 10](2):305-6 (1991)). Thehuman sequence had an overall identity to the 1D9 V_(K) region of 79.3%and 85.0% within the FRs alone. When measured with respect tosimilarity, these values increase to 83.99% overall and 87.5% within theFRs alone. In addition, key FR amino acids were more conservativelypreserved in HF-21/28 V_(K) region than in the other candidate humankappa light chain variable regions. Consequently, the HF-21/28 kappalight chain variable region FR was selected as the human acceptorsequence for the humanization of the 1D9 antibody kappa light chainvariable region.

[0262] Unfortunately, the very last residue in FR4 (at position 107,according to the Kabat numbering system) of the human HF-21/28 V_(K)region was not defined by the Kabat database or the authors whooriginally isolated this variable region sequence. Therefore, it wasdecided to insert the most commonly found amino acid at this position inthe variable region sequences described by Kabat human kappa light chainsubgroup κ-II (Kabat et al., Sequences of proteins of immunologicalinterest, Fifth edition, U.S. Department of Health and Human Services,U.S. Government Printing Office (1991)). Accordingly, lysine was addedat position 107 in FR4 based upon an analysis of the Kabat databasewhich found that 85.7% of the sequences in Kabat human kappa light chainsubgroup _(κ)-II had a lysine at this position. This then became thebasis of the first humanized version of the 1D9 kappa lightchain(1D9RK_(A)), which essentially comprised the CDR's of the 1D9 V_(K)region and the FRs of HF-21/28 V_(K) region. FIGS. 19A-19C define theamino acid sequence of this first CDR-grafted version of the humanised1D9 V_(K) region.

[0263] The next step in the design process was to study the amino acidsequences of the human acceptor HF-21/28 V_(K) region FRs to determineif any of these amino acid residues were likely to adversely influencebinding to antigen. This could be caused directly through interactionswith antigen, or indirectly by altering the confirmation or orientationof the CDR loops. This was a difficult process which was only madepossible through the availability of a model of the 1D9 variableregions, i.e., both the V_(K) and the V_(H) regions. Nevertheless, anyamino acids in the mouse 1D9 FRs that did appear to affect antigenbinding were then considered for conversion in the humanized 1D9antibody. In deciding which murine residues to conserve the followingpoints were addressed:

[0264] It was of great importance that the canonical structures of thehypervariable loops (Chothia and Lesk, J. Mol. Biol. 197:901 (1987);Chothia et al., Nature 34:877 (1989); Tramontano et al., J. Mol. Biol.215:175 (1990); and Chothia et al., J. Mol. Biol. 227:799 (1992)) wereconserved. Consequently, it was crucial to conserve in the humanized 1D9variable regions all the mouse FR residues that were part of thesecanonical structures (FIGS. 9 and 10).

[0265] The sequences of the 1D9 antibody variable regions were comparedto similar sequences from other mouse antibodies to identify unusual orrare residues which may have indicated an important role in antigenbinding. This was then investigated using the mouse model of the 1D9variable region genes.

[0266] A direct analysis of the model was also made to try and predictwhether any of the other mouse FR residues not present in the humanizedFRs could influence antigen binding in some way.

[0267] Comparisons of the individual human acceptor sequences for thekappa light chain and heavy chain variable regions to the consensussequence of human variable regions subgroups to which the acceptorsequences belonged were also made. The identification of anyidiosyncratic amino acids in the human donor sequences was important, asthese could have adversely affected antigen binding.

[0268] Since the human light and heavy chain variable regions selectedwould be derived from two different human antibodies, a careful analysisof the interdomain packing residues of both the donor and the acceptorkappa light chain variable regions should be carried out (Chothia etal., J. Mol. Biol. 186:651 (1985)). This was because any mispacking inthis region could have had a dramatic affect upon antigen binding,irrespective of the conformation of the CDR loop structures of thehumanized 1D9 antibody.

[0269] Although there were 12 amino acid differences between the FRs ofthe donor mouse 1D9 V_(K) region and the acceptor human HF-21/28 V_(K)region, only two mouse residues were considered sufficiently importantfor binding affinity to preserve them in the humanised FRs. The first ofthe FR changes that were introduced into 1D⁹RK_(B) was located atposition 36. This residue is a Vernier residue (Foote and Winter, J.Mol. Biol. 224:487 (1992)) and is predicted to be a key structuredetermining residue for L1 loop structure as defined by Chothia and hiscoworkers (Chothia and Lesk, J. Mol. Biol. 197:901 (1987); Chothia etal., Nature 34:877 (1989); Tramontano et al., J. Mol. Biol. 215:175(1990); and Chothia et al., J. Mol. Biol. 227:799 (1992)). Although bothresidues are hydrophobic, the human Phe is bulkier at this position, andX-ray structures with Leu and Phe at this position show that if Phe ispresent steric hindrance causes the side chain at 34Asn to point in theopposite direction. Thus, it was considered critical for the successfulhumanization of the 1D9 kappa light chain.

[0270] The second change incorporated into the 1D⁹RKB humanized versionwas at residue 37, i.e., Gln37Leu. Although this was a conservativechange it occurred in a highly conserved region at the base of CDR1. Itwas thought that by preserving this murine Leu residue in this version,alongside the murine Leu residue at position 36, the affinity of thehumanised antibody could be preserved.

[0271] Two other versions of the humanised V_(K) region were alsoconsidered for construction to explore the structural and bindingaffinity consequences of manipulating the FRs of the humanised 1D9antibody. 1D9RK_(C) was essentially identical to 1D⁹RK_(B), except forthe mutation Gln100Gly. There is a dramatic difference in molecule bulkbetween these two residues, and thus this version was made to explorethe consequences of this change to the FRs of the reshaped human kappalight chain on antibody structure and overall antibody affinity.1D9RK_(D) contained the modifications described in 1D9RK_(C) and, inaddition, contained the FR change Gln17His. Although Gln and His aresimilar in size and both are weakly polar, the mouse residue (His) atthis position is extremely rare amongst all mouse V_(K) sequences (0.07%overall, but has not been seen in mouse Kabat subgroup II sequences) andhas never been seen in any human V_(K) sequences. Conversely, the Glnresidue is more commonly seen at this position in both mouse (16.16%overall and 6.12% in mouse Kabat subgroup II sequences) and human (5.00%overall and 39.7% in human Kabat subgroup II sequences) sequences. Thus,the simple rarity of the His at this position suggests that it may beimportant for binding, although there is no clear evidence to supportthis from the molecular modeling data. A description of the amino acidsequences of all the humanised 1D9 antibody V_(K) region variantsproposed above are given in FIG. 11.

[0272] Design of Humanised 1D9 V_(H) Antibody Variants

[0273] Once again, the first step in the design of the humanised V_(H)region of the mouse 1D9 antibody was the selection of the acceptor humanheavy chain variable region that would serve as the basis of thehumanised 1D9 V_(H) region. When the 1D9 V_(H) region was initiallycompared to the consensus sequences of the three human heavy chainvariable region subgroups it was found to be most similar to theconsensus sequence for the human heavy chain subgroup III, with a69.231% identity overall and a 78.161% identity between the FRs alone.When measured with respect to similarity, these values increased to74.359% overall and to 82.759% within the FRs alone.

[0274] The mouse 1D9 V_(H) region was then compared to all the recordedexamples of individual sequences of human variable regions publiclyavailable. FIGS. 17A-B show the best 24 matches to the mouse 1D9 V_(H)region which were identified through this analysis. Overall the searchalgorithm selected the human V_(H) region from antibody 4B4′CL (Kabatdata base ID number 000490; Sanz et al., Journal of Immunology. 142:883(1989)) as the best match to the mouse 1D9 V_(H) region. The V_(H)region of this clone had an overall identity to the 1D9 V_(H) region of67.2%, a value which increased to 80.95% when the FRs alone werecompared (FIGS. 18A-B). When measured with respect to similarity, thesevalues increased to 69.66% overall and to 84.52% within the FRs alone.Thus, although once again not the most homologous of the potential humanacceptor V_(H) sequences, this human FR became the basis of thehumanised version of the 1D9 heavy chain.

[0275] The next step in the design process was to study the amino acidsequences of the human acceptor 4B4′CL V_(H) region FRs to determine ifany of these amino acid residues were likely to adversely affect bindingto antigen. Once again the molecular models built by OML were importantin this design process, from which a number of amino acids in the murine1D9 V_(H) region FRs were identified for conversion in the first(1D9RH_(A)) and subsequent versions of the humanised 1D9 antibody (FIG.12 and FIGS. 20A-C). There were 16 amino acid differences between theFRs of the donor mouse 1D9 and the acceptor human 4B4′CL V_(H) regions,and up to 5 murine residues were considered for conservation in thehumanised FRs (FIG. 12).

[0276] 1D⁹RHA consisted of the CDRs of the murine 1D9 antibody V_(H)region genetically inserted into the FRs of the human 4B4′CL antibodyV_(H) region. 1D9RH_(B) was identical to version 1D⁹RH_(A) apart fromtwo FR1 mutations, Thr28Ser and Asn30Ser. These changes were madebecause they represented Vernier amino acids as defined by Foote andWinter (J. Mol. Biol. 224:487 (1992)), which were thought to be criticalfor H1 loop conformation. Residues 27-30 are considered part of the H1loop itself and so are even more critical to the correct conformationand orientation of this loop, justifying their conservation even morestrongly. Thus, these two residues represented the sum of the changesmade to the FRs of the human 4B4′CL V_(H) sequence in 1D9RH_(B).1D9RH_(C) was identical to version 1D9RH_(B) except that it containedtwo further changes at positions Gly49Ala and Phe67Tyr. The Gly49Ala wasa conservative change. However, residue position 49 has been identifiedas a Vernier residue (Foote and Winter, J. Mol. Biol. 224:487 (1992)),important for H2 hypervariable loop structure, so it was decided toconserve the murine Ala residue in this version. Residue position 67 wasalso a Vernier residue position, identifying it as important formaintaining CDR loop conformation. Tyr is very rarely seen in humanV_(H) sequences (0.08% overall) and has not previously been found inmurine V_(H) regions at this position. Consequently, it must have arisenthrough somatic mutation. Thus, given its location close to CDR2according to the molecular model and its Vernier residue status, it wasdecided to conserve the murine Tyr residue at this position. 1D9RH_(D)was identical to 1D9RH_(C) except for a Thr93Val mutation. This residuehad been identified as important as both a V_(H/)V_(K) packing residue(Chothia et al., J. Mol. Biol. 186:651 (1985)). Moreover, its buriedposition between CDR loops H1 and H3, according to the molecular model,supported the decision to conserve the murine Val residue at thisposition. A description of the amino acid sequences of all the humanisedV_(H) region variants described above are given in FIG. 12.

[0277] While this invention has been particularly shown and describedwith references to preferred embodiments thereof, it will be understoodby those skilled in the art that various changes in form and details maybe made therein without departing from the scope of the inventionencompassed by the appended claims.

1 106 1 22 DNA Artificial Sequence Primer sequence 1 tgagacaagccacaagctga ac 22 2 22 DNA Artificial Sequence Primer sequence 2tctgtattag tacacacagc cc 22 3 24 DNA Artificial Sequence Primer sequence3 atgctgtcca catctcgttc tcgg 24 4 27 DNA Artificial Sequence Primersequence 4 ttataaacca gccgagactt cctgctc 27 5 24 PRT Artificial SequenceCD5 signal peptide leader sequence 5 Met Pro Met Gly Ser Leu Gln Pro LeuAla Thr Leu Tyr Leu Leu Gly 1 5 10 15 Met Leu Val Ala Ser Val Leu Ala 206 60 DNA Artificial Sequence Primer 6 ggggatccag aaaccatgcc catggggtctctgcaaccgc tggccacctt gtacctgctg 60 7 65 DNA Artificial Sequence Primer7 gccaccttgt acctgctggg gatgctggtc gcttccgtgc tagcgatgct gtccacatct 60cgttc 65 8 18 DNA Artificial Sequence Primer 8 gacgaccagc atgttgcc 18 9112 PRT Mus musculus 9 Asp Val Val Met Thr Gln Thr Pro Leu Thr Leu SerVal Thr Val Gly 1 5 10 15 His Pro Ala Ser Ile Ser Cys Lys Ser Ser GlnSer Leu Leu Asp Ser 20 25 30 Asp Gly Lys Thr Phe Leu Asn Trp Leu Leu GlnArg Pro Gly Gln Ser 35 40 45 Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys LeuAsp Ser Gly Val Pro 50 55 60 Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr AspPhe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly ValTyr Tyr Cys Trp Gln Gly 85 90 95 Thr His Phe Pro Tyr Thr Phe Gly Gly GlyThr Lys Leu Glu Ile Lys 100 105 110 10 117 PRT Mus musculus 10 Glu ValGln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Lys Gly 1 5 10 15 SerLeu Lys Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Asn Ala Tyr 20 25 30 AlaMet Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 AlaArg Ile Arg Thr Lys Asn Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 SerVal Lys Asp Arg Tyr Thr Ile Ser Arg Asp Asp Ser Glu Ser Met 65 70 75 80Leu Phe Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr 85 90 95Tyr Cys Val Thr Phe Tyr Gly Asn Gly Val Trp Gly Thr Gly Thr Thr 100 105110 Val Thr Val Ser Ser 115 11 111 PRT Homo sapiens 11 Asp Val Val MetThr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro AlaSer Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asp Gly AsnThr Tyr Leu Asn Trp Phe Gln Gln Arg Pro Gly Gln Ser 35 40 45 Pro Arg ArgLeu Ile Tyr Lys Val Ser Asn Arg Asp Ser Gly Val Pro 50 55 60 Asp Arg PheSer Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser ArgVal Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Gly 85 90 95 Thr HisTrp Pro Phe Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile 100 105 110 12 112PRT Artificial Sequence Humanized sequence 12 Asp Val Val Met Thr GlnSer Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser IleSer Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser 20 25 30 Asp Gly Lys Thr PheLeu Asn Trp Phe Gln Gln Arg Pro Gly Gln Ser 35 40 45 Pro Arg Arg Leu IleTyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser GlySer Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val GluAla Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly 85 90 95 Thr His Phe ProTyr Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105 110 13 112 PRTArtificial Sequence Humanized sequence 13 Asp Val Val Met Thr Gln SerPro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile SerCys Lys Ser Ser Gln Ser Leu Leu Asp Ser 20 25 30 Asp Gly Lys Thr Phe LeuAsn Trp Leu Leu Gln Arg Pro Gly Gln Ser 35 40 45 Pro Arg Arg Leu Ile TyrLeu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly SerGly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu AlaGlu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly 85 90 95 Thr His Phe Pro TyrThr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105 110 14 112 PRTArtificial Sequence Humanized sequence 14 Asp Val Val Met Thr Gln SerPro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile SerCys Lys Ser Ser Gln Ser Leu Leu Asp Ser 20 25 30 Asp Gly Lys Thr Phe LeuAsn Trp Leu Leu Gln Arg Pro Gly Gln Ser 35 40 45 Pro Arg Arg Leu Ile TyrLeu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly SerGly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu AlaGlu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly 85 90 95 Thr His Phe Pro TyrThr Phe Gly Gly Gly Thr Arg Leu Glu Ile Lys 100 105 110 15 112 PRTArtificial Sequence Humanized sequence 15 Asp Val Val Met Thr Gln SerPro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 His Pro Ala Ser Ile SerCys Lys Ser Ser Gln Ser Leu Leu Asp Ser 20 25 30 Asp Gly Lys Thr Phe LeuAsn Trp Leu Leu Gln Arg Pro Gly Gln Ser 35 40 45 Pro Arg Arg Leu Ile TyrLeu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly SerGly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu AlaGlu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly 85 90 95 Thr His Phe Pro TyrThr Phe Gly Gly Gly Thr Arg Leu Glu Ile Lys 100 105 110 16 119 PRT Homosapiens 16 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro GlyGly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe SerAsn Ala 20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu GluTrp Val 35 40 45 Gly Arg Ile Lys Ser Lys Thr Asp Gly Gly Thr Thr Asp TyrAla Ala 50 55 60 Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser LysAsn Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp ThrAla Val Tyr 85 90 95 Tyr Cys Thr Thr Asp Ser Leu Pro Pro His Arg Val TrpGly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 17 117 PRTArtificial Sequence Humanized sequence 17 Glu Val Gln Leu Val Glu SerGly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser CysAla Ala Ser Gly Phe Thr Phe Ser Ala Tyr 20 25 30 Ala Met Asn Trp Val ArgGln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Arg Thr LysAsn Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg PheThr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln MetAsn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Thr Thr PheTyr Gly Asn Gly Val Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val SerSer 115 18 117 PRT Artificial Sequence Humanized sequence 18 Glu Val GlnLeu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser LeuArg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Asn Ala Tyr 20 25 30 Ala MetAsn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly ArgIle Arg Thr Lys Asn Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser ValLys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 LeuTyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 TyrCys Thr Thr Phe Tyr Gly Asn Gly Val Trp Gly Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ser 115 19 117 PRT Artificial Sequence Humanizedsequence 19 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro GlyGly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe AsnAla Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu GluTrp Val 35 40 45 Ala Arg Ile Arg Thr Lys Asn Asn Asn Tyr Ala Thr Tyr TyrAla Asp 50 55 60 Ser Val Lys Asp Arg Tyr Thr Ile Ser Arg Asp Asp Ser LysAsn Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp ThrAla Val Tyr 85 90 95 Tyr Cys Thr Thr Phe Tyr Gly Asn Gly Val Trp Gly GlnGly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 20 117 PRT ArtificialSequence Humanized sequence 20 Glu Val Gln Leu Val Glu Ser Gly Gly GlyLeu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala SerGly Phe Ser Phe Asn Ala Tyr 20 25 30 Ala Met Asn Trp Val Arg Gln Ala ProGly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Arg Thr Lys Asn Asn AsnTyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser Val Lys Asp Arg Tyr Thr Ile SerArg Asp Asp Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser LeuLys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Val Thr Phe Tyr Gly AsnGly Val Trp Gly Gln Gly Thr Leu 100 105 110 Val Thr Val Ser Ser 115 21100 PRT Mus musculus 21 Asp Val Val Met Thr Gln Thr Pro Leu Thr Leu SerVal Thr Val Gly 1 5 10 15 His Pro Ala Ser Ile Ser Cys Lys Ser Ser GlnSer Leu Leu Asp Ser 20 25 30 Asp Gly Lys Thr Phe Leu Asn Trp Leu Leu GlnArg Pro Gly Gln Ser 35 40 45 Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys LeuAsp Ser Gly Val Pro 50 55 60 Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr AspPhe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly ValTyr Tyr Cys Trp Gln Gly 85 90 95 Thr His Phe Pro 100 22 100 PRT Musmusculus 22 Asp Val Val Met Thr Gln Thr Pro Leu Thr Leu Ser Val Thr IleGly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu LeuAsp Ser 20 25 30 Asp Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro GlyGln Ser 35 40 45 Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser GlyVal Pro 50 55 60 Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr LeuLys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr CysTrp Gln Gly 85 90 95 Thr His Phe Pro 100 23 100 PRT Mus musculus 23 AspVal Val Met Thr Gln Thr Pro Leu Thr Leu Ser Val Thr Ile Gly 1 5 10 15Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser 20 25 30Asn Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser 35 40 45Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 7580 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Val Gln Gly 85 9095 Thr His Phe Pro 100 24 100 PRT Mus musculus VARIANT (1)...(100) Xaa =Any Amino Acid 24 Asp Val Val Met Thr Gln Xaa Leu His Ser Leu Ser ValThr Ile Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln SerLeu Leu Tyr Ser 20 25 30 Asn Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln ArgPro Val Gln Pro 35 40 45 Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu TyrSer Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp PheThr Leu Lys Ile 65 70 75 80 Ser Arg Val Xaa Pro Glu Asp Leu Gly Val TyrXaa Cys Met Gln Asp 85 90 95 Thr His Phe Pro 100 25 100 PRT Mus musculus25 Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 510 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 2025 30 Asn Gly Asn Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser 3540 45 Pro Lys Leu Leu Ile Tyr Arg Val Ser Asn Arg Phe Ser Gly Val Pro 5055 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 6570 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Phe Gln Gly85 90 95 Thr His Val Pro 100 26 100 PRT Mus musculus 26 Asp Val Val MetThr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln AlaSer Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser 20 25 30 Asn Gly AsnThr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys LeuLeu Ile Tyr Lys Val Ser Asn Arg Leu Ser Gly Val Pro 50 55 60 Asp Arg PheSer Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser ArgVal Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly 85 90 95 Ser HisVal Pro 100 27 100 PRT Mus musculus 27 Asp Val Val Met Thr Gln Thr ProLeu Ser Leu Pro Val Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser CysArg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu HisTrp Tyr Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr LysVal Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser GlySer Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala GluAsp Leu Gly Val Tyr Phe Cys Ser Gln Ser 85 90 95 Thr His Val Pro 100 28100 PRT Mus musculus 28 Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu ProVal Ser Leu Gly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser GlnSer Ile Val His Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu GlnLys Pro Gly Gln Ser 35 40 45 Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn ArgPhe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr AspPhe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly ValTyr Tyr Cys Phe Gln Gly 85 90 95 Ser His Val Pro 100 29 100 PRT Musmusculus 29 Asp Ala Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser LeuGly 1 5 10 15 Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu GluAsn Ser 20 25 30 Asn Gly Asn Thr Tyr Leu Asn Trp Tyr Leu Gln Lys Pro GlyGln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Arg Val Ser Asn Arg Phe Ser GlyVal Leu 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr LeuLys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe CysLeu Gln Val 85 90 95 Thr His Val Pro 100 30 100 PRT Mus musculus 30 AspVal Leu Leu Thr Gln Thr Pro Leu Phe Leu Pro Val Ser Leu Gly 1 5 10 15Asp Gln Ala Ser Ile Ser Cys Ser Ser Ser Gln Ser Leu Val His Ser 20 25 30Asn Gly Asn Tyr Tyr Leu Glu Trp His Leu Gln Lys Ser Gly Gln Ser 35 40 45Leu Gln Leu Leu Ile Tyr Glu Val Ser Lys Arg His Ser Gly Val Pro 50 55 60Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 7580 Ser Arg Val Glu Pro Glu Asp Leu Gly Val Tyr Tyr Cys Phe Gln Gly 85 9095 Thr His Leu Pro 100 31 100 PRT Mus musculus 31 Asp Ile Val Met ThrGln Ala Ala Phe Ser Asn Pro Val Thr Leu Gly 1 5 10 15 Thr Ser Ala SerIle Ser Cys Arg Ser Ser Lys Ser Leu Leu His Ser 20 25 30 Ser Gly Asn ThrTyr Leu Tyr Trp Phe Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu LeuIle Tyr Tyr Ile Ser Asn Leu Ala Ser Gly Val Pro 50 55 60 Asp Arg Phe SerGly Ser Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile 65 70 75 80 Ser Arg ValGlu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Gly 85 90 95 Leu Glu TyrPro 100 32 100 PRT Mus musculus 32 Asp Ile Val Ile Thr Gln Asp Glu LeuSer Asn Pro Val Thr Ser Gly 1 5 10 15 Glu Ser Val Ser Ile Ser Cys ArgSer Ser Lys Ser Leu Leu Tyr Lys 20 25 30 Asp Gly Lys Thr Tyr Leu Asn TrpPhe Leu Gln Arg Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Leu MetSer Thr Arg Ala Ser Gly Val Ser 50 55 60 Asp Arg Phe Ser Gly Ser Gly SerGly Thr Asp Phe Thr Leu Glu Ile 65 70 75 80 Ser Arg Val Lys Ala Glu AspVal Gly Val Tyr Tyr Cys Gln Gln Leu 85 90 95 Val Glu Tyr Pro 100 33 100PRT Mus musculus 33 Asp Ile Val Met Thr Gln Ala Ala Phe Ser Asn Pro ValThr Leu Gly 1 5 10 15 Thr Ser Ala Ser Ile Ser Cys Arg Ser Ser Lys SerLeu Leu His Ser 20 25 30 Asn Gly Ile Thr Tyr Leu Tyr Trp Tyr Leu Gln LysPro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu AlaSer Gly Val Pro 50 55 60 Asp Arg Phe Ser Ser Ser Gly Ser Gly Thr Asp PheThr Leu Arg Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val TyrTyr Cys Ala Gln Asn 85 90 95 Leu Glu Leu Pro 100 34 101 PRT Mus musculus34 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Lys Gly 1 510 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe Asn Ala Tyr 2025 30 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 3540 45 Ala Arg Ile Arg Thr Lys Asn Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 5055 60 Ser Val Lys Asp Arg Tyr Thr Ile Ser Arg Asp Asp Ser Glu Ser Met 6570 75 80 Leu Phe Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr85 90 95 Tyr Cys Val Thr Phe 100 35 100 PRT Mus musculus 35 Glu Val GlnLeu Val Glu Val Trp Trp Arg Met Val Gln Pro Lys Gly 1 5 10 15 Ser LeuLys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Ala MetAsn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala ArgIle Arg Ser Lys Ser Ser Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60 Ser ValLys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Gln Ser Met 65 70 75 80 LeuTyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr 85 90 95 TyrCys Val Ile 100 36 100 PRT Mus musculus 36 Glu Val Lys Leu Glu Glu SerGly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Met Lys Leu Ser CysVal Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Trp Met Ser Trp Val ArgGln Ser Pro Glu Lys Gly Leu Glu Trp Val 35 40 45 Ala Gln Ile Arg Leu LysSer Asp Asn Tyr Ala Thr His Tyr Ala Glu 50 55 60 Ser Val Lys Gly Arg PheThr Ile Ser Arg Asp Asp Ser Lys Ser Ser 65 70 75 80 Val Tyr Leu Gln MetAsn Asn Leu Arg Ala Glu Asp Thr Gly Ile Tyr 85 90 95 Tyr Cys Thr Gly 10037 100 PRT Mus musculus 37 Glu Val Lys Leu Val Glu Ser Gly Gly Gly LeuVal Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Thr Ser GlyPhe Thr Phe Thr Asp Tyr 20 25 30 Tyr Met Ser Trp Val Arg Gln Pro Pro GlyLys Ala Leu Glu Trp Leu 35 40 45 Gly Phe Ile Arg Asn Lys Ala Asn Gly TyrThr Thr Glu Tyr Ser Ala 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser ArgAsp Asn Ser Gln Ser Ile 65 70 75 80 Leu Tyr Leu Gln Met Asn Thr Leu ArgAla Glu Asp Ser Ala Thr Tyr 85 90 95 Tyr Cys Ala Arg 100 38 98 PRT Musmusculus 38 Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro GlyGly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe SerSer Tyr 20 25 30 Thr Met Ser Trp Val Arg Gln Ser Pro Glu Lys Arg Leu GluTrp Val 35 40 45 Ala Thr Ile Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr Pro AspSer Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn ThrLeu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr Ala MetTyr Tyr Cys 85 90 95 Thr Arg 39 98 PRT Mus musculus 39 Asp Val Lys LeuVal Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu LysLeu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Thr Met SerTrp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr IleSer Ser Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val 50 55 60 Lys Gly ArgPhe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu GlnMet Ser Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Thr Arg40 98 PRT Mus musculus VARIANT (1)...(98) Xaa = Any Amino Acid 40 GluLeu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15Ser Arg Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30Ala Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45Ala Ala Ile Ser Thr Asp Gly Ser Phe Ile Tyr Xaa Pro Asp Thr Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Phe 65 70 7580 Leu Gln Met Ser Ser Leu Arg Tyr Glu Asp Thr Ala Met Tyr Tyr Cys 85 9095 Leu Arg 41 98 PRT Mus musculus 41 Glu Val Lys Leu Val Glu Ser Gly GlyGly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala ThrSer Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Tyr Met Tyr Trp Val Arg Gln ThrPro Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Tyr Ile Ser Asn Gly Gly GlySer Thr Tyr Tyr Pro Asp Thr Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser ArgAsp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Ser Arg Leu LysSer Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg 42 101 PRT Musmusculus 42 Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro GlyAla 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ser Ser Gly Phe Thr Phe ThrAsp Tyr 20 25 30 Tyr Met Asn Trp Val His Arg Pro Pro Gly Lys Pro Leu GluTrp Leu 35 40 45 Ala Leu Ile Arg Asn Lys Ala Asn Gly Tyr Ile Thr Glu TyrSer Ala 50 55 60 Ser Met Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser GlnSer Ile 65 70 75 80 Leu Tyr Leu Gln Met Asn Thr Leu Ser Thr Glu Asp SerAla Thr Tyr 85 90 95 Tyr Cys Ala Arg Asp 100 43 100 PRT Mus musculus 43Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 1015 Ser Leu Arg Leu Ser Cys Ala Thr Ser Gly Phe Thr Phe Ser Asp Phe 20 2530 Tyr Met Glu Trp Val Arg Gln Pro Pro Gly Lys Arg Leu Glu Trp Ile 35 4045 Ala Ala Ser Arg Asn Lys Ala Asn Asp Tyr Thr Thr Glu Tyr Ser Ala 50 5560 Ser Val Lys Gly Arg Phe Ile Val Ser Arg Asp Thr Ser Gln Ser Ile 65 7075 80 Leu Tyr Leu Gln Met Asn Ala Leu Arg Ala Glu Asp Thr Ala Ile Tyr 8590 95 Tyr Cys Ala Arg 100 44 98 PRT Mus musculus 44 Glu Val Met Leu ValGlu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys LeuSer Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Thr Met Ser TrpVal Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Ile SerSer Gly Gly Gly Asn Thr Tyr Tyr Pro Asp Ser Val 50 55 60 Lys Gly Arg PheThr Ile Ser Arg Asp Asn Ala Lys Asn Asn Leu Tyr 65 70 75 80 Leu Gln MetSer Ser Leu Arg Ser Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Arg 4598 PRT Mus musculus 45 Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu ValLys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Thr Ser Gly PheThr Phe Ser Ser Tyr 20 25 30 Gly Met Ser Trp Val Arg Gln Thr Pro Glu LysArg Leu Glu Trp Val 35 40 45 Ala Thr Ile Ser Gly Gly Gly Ser Tyr Thr TyrTyr Pro Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn AlaLys Asn Asn Leu Tyr 65 70 75 80 Leu Gln Met Ser Ser Leu Arg Ser Glu AspThr Ala Leu Tyr Tyr Cys 85 90 95 Ala Arg 46 101 PRT Mus musculus 46 GluVal Lys Leu Met Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Ala 1 5 10 15Ser Leu Arg Leu Ser Cys Glu Ala Ser Gly Phe Thr Phe Thr Asp Tyr 20 25 30Tyr Met Ser Trp Val Arg Gln Leu Pro Arg Lys Ser Pro Glu Trp Leu 35 40 45Ala Leu Ile Arg Asn Lys Ala Asn Gly Tyr Thr Thr Glu Tyr Ser Ala 50 55 60Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Gln Asn Ile 65 70 7580 Leu Tyr Leu Gln Met Asn Thr Leu Arg Ala Glu Ala Ser Ala Thr Tyr 85 9095 Tyr Cys Ala Lys Asp 100 47 98 PRT Mus musculus 47 Glu Val Lys Leu LeuGlu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys LeuSer Cys Ala Ala Ser Gly Phe Asp Phe Ser Arg Tyr 20 25 30 Trp Met Ser TrpVal Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile AsnPro Asp Ser Ser Thr Ile Asn Tyr Thr Pro Ser Leu 50 55 60 Lys Asp Lys PheIle Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln MetSer Lys Val Arg Ser Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Arg 4889 PRT Mus musculus 48 Gly Leu Val Gln Pro Gly Gly Ser Leu Lys Leu SerCys Ala Ala Ser 1 5 10 15 Gly Phe Thr Phe Ser Ser Tyr Gly Met Ser TrpVal Arg Gln Thr Pro 20 25 30 Asp Lys Arg Leu Glu Leu Val Ala Thr Ile AsnSer Asn Gly Gly Ser 35 40 45 Thr Tyr Tyr Pro Asp Ser Val Lys Gly Arg PheThr Ile Ser Arg Asp 50 55 60 Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met SerSer Leu Lys Ser Glu 65 70 75 80 Asp Thr Ala Met Tyr Tyr Cys Ala Arg 8549 89 PRT Mus musculus 49 Gly Leu Val Lys Pro Gly Gly Ser Leu Lys LeuSer Cys Ala Ala Ser 1 5 10 15 Gly Phe Thr Phe Ser Ser Tyr Ala Met SerTrp Val Arg Gln Thr Pro 20 25 30 Glu Lys Arg Leu Glu Trp Val Ala Thr IleSer Ser Gly Gly Ser Tyr 35 40 45 Thr Tyr Tyr Pro Asp Ser Val Lys Gly ArgPhe Thr Ile Ser Arg Asp 50 55 60 Asn Ala Lys Asn Thr Leu Tyr Leu Gln MetSer Ser Leu Arg Ser Glu 65 70 75 80 Asp Thr Ala Met Tyr Tyr Cys Ala Arg85 50 89 PRT Mus musculus 50 Gly Leu Val Gln Pro Gly Gly Ser Arg Lys LeuSer Cys Ala Ala Ser 1 5 10 15 Gly Phe Thr Phe Ser Ser Phe Gly Met HisTrp Val Arg Gln Ala Pro 20 25 30 Glu Lys Gly Leu Glu Trp Val Ala Tyr IleSer Ser Gly Ser Ser Thr 35 40 45 Ile Tyr Tyr Ala Asp Thr Val Lys Gly ArgPhe Thr Ile Ser Arg Asp 50 55 60 Asn Pro Lys Asn Thr Leu Phe Leu Gln MetThr Ser Leu Arg Ser Glu 65 70 75 80 Asp Thr Ala Met Tyr Tyr Cys Ala Arg85 51 88 PRT Mus musculus 51 Gly Leu Val Lys Pro Gly Gly Ser Leu Lys LeuSer Cys Ala Ala Ser 1 5 10 15 Gly Phe Thr Phe Ser Ser Tyr Ala Met SerTrp Val Arg Gln Thr Pro 20 25 30 Glu Lys Arg Leu Glu Trp Val Ala Ser IleSer Ser Gly Gly Ser Thr 35 40 45 Tyr Tyr Pro Asp Ser Val Lys Gly Arg PheThr Ile Ser Arg Asp Asn 50 55 60 Ala Arg Asn Ile Leu Tyr Leu Gln Met SerSer Leu Arg Ser Glu Asp 65 70 75 80 Thr Ala Met Tyr Tyr Cys Ala Arg 8552 98 PRT Mus musculus 52 Glu Val Lys Leu Leu Glu Ser Gly Gly Gly LeuVal Gln Pro Gly Gly 1 5 10 15 Ser Leu Asn Leu Ser Cys Ala Ala Ser GlyPhe Asp Phe Ser Arg Tyr 20 25 30 Trp Met Ser Trp Ala Arg Gln Ala Pro GlyLys Gly Gln Glu Trp Ile 35 40 45 Gly Glu Ile Asn Pro Gly Ser Ser Thr IleAsn Tyr Thr Pro Ser Leu 50 55 60 Lys Asp Lys Phe Ile Ile Ser Arg Asp AsnAla Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Ser Lys Val Arg Ser GluAsp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Arg 53 87 PRT Mus musculus 53Val Lys Pro Gly Gly Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe 1 5 1015 Thr Phe Ser Ser Tyr Thr Met Ser Trp Val Arg Gln Thr Pro Glu Lys 20 2530 Arg Leu Glu Trp Val Ala Tyr Ile Ser Asn Gly Gly Gly Ser Thr Tyr 35 4045 Tyr Pro Asp Thr Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala 50 5560 Lys Asn Thr Leu Tyr Leu Gln Met Ser Ser Leu Lys Ser Glu Asp Thr 65 7075 80 Ala Met Tyr Tyr Cys Ala Arg 85 54 112 PRT Homo sapiens 54 Asp IleGln Leu Thr Gln Ser Pro Leu Thr Leu Ser Val Thr Ile Gly 1 5 10 15 GlnPro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser 20 25 30 AspGly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser 35 40 45 ProLys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60 AspArg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80Ser Arg Val Glu Ala Asp Asp Leu Gly Val Tyr Tyr Cys Trp Gln Gly 85 90 95Thr His Phe Pro Gln Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105110 55 112 PRT Homo sapiens 55 Asp Val Val Leu Thr Gln Ser Pro Leu SerLeu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg SerAsp Gln Ser Leu Val Tyr Ser 20 25 30 Asp Gly Lys Thr Tyr Leu Asn Trp TyrGln Gln Arg Pro Gly Gln Ser 35 40 45 Pro Arg Arg Leu Ile Tyr Lys Val SerAsn Arg Asp Ser Gly Val Pro 50 55 60 Asp Arg Phe Thr Gly Ser Gly Ser GlyThr Asp Phe Thr Leu Glu Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp ValGly Val Tyr Tyr Cys Met Gln Gly 85 90 95 Thr His Trp Pro Gly Thr Phe GlyGln Gly Thr Lys Val Glu Ile Lys 100 105 110 56 112 PRT Homo sapiens 56Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 1015 Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val Tyr Ser 20 2530 Asp Gly Asn Thr Tyr Leu Asn Trp Phe Gln Gln Arg Pro Gly Gln Ser 35 4045 Pro Arg Arg Leu Ile Tyr Lys Val Ser Asn Arg Asp Ser Gly Val Pro 50 5560 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 7075 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Gly 8590 95 Thr His Trp Ser Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys100 105 110 57 112 PRT Homo sapiens 57 Asp Val Val Val Thr Gln Ser ProLeu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser CysArg Ser Ser Leu Ser Leu Val Asp Ser 20 25 30 Asp Gly Asn Thr Tyr Leu AsnTrp Phe Leu Gln Arg Pro Gly Gln Ser 35 40 45 Pro Arg Arg Leu Ile Tyr GlnLeu Ser Ser Arg Asp Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser GlySer Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala GluAsp Val Gly Val Tyr Tyr Cys Met Gln Gly 85 90 95 Thr His Trp Pro Ile ThrPhe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105 110 58 112 PRT Homosapiens 58 Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr LeuGly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Gly Leu ValTyr Ser 20 25 30 Asp Gly Asp Thr Tyr Leu Asn Trp Phe Gln Gln Arg Pro GlyGln Ser 35 40 45 Pro Arg Arg Leu Ile Tyr Lys Val Ser Asn Arg Asp Ser GlyVal Pro 50 55 60 Asp Arg Phe Ser Gly Gly Gly Ser Gly Thr Asp Phe Thr LeuLys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr CysMet Gln Gly 85 90 95 Thr His Trp Pro Tyr Thr Phe Gly Gln Gly Thr Lys LeuGlu Ile Lys 100 105 110 59 111 PRT Homo sapiens 59 Asp Val Val Met ThrGln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala SerIle Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 Asp Gly Asn ThrTyr Leu Asn Trp Phe Gln Gln Arg Pro Gly Gln Ser 35 40 45 Pro Arg Arg LeuIle Tyr Lys Val Ser Asn Arg Asp Ser Gly Val Pro 50 55 60 Asp Arg Phe SerGly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg ValGlu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Gly 85 90 95 Thr His TrpPro Phe Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile 100 105 110 60 112 PRTHomo sapiens 60 Ala Glu Glu Leu Thr Gln Ser Pro Leu Ser Leu Pro Val ThrLeu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser LeuLeu Leu Ser 20 25 30 Asp Gly Asp Thr Tyr Leu Asn Trp Tyr Gln Gln Arg ProGly Gln Ser 35 40 45 Pro Arg Arg Leu Ile Tyr Lys Val Ser Asn Arg Asp SerGly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe ThrLeu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr TyrCys Met Gln Gly 85 90 95 Ala His Trp Pro Tyr Thr Phe Gly Gln Gly Thr LysLeu Glu Ile Lys 100 105 110 61 112 PRT Homo sapiens 61 Asp Val Val LeuThr Gln Ser Pro Leu Ser Leu Ser Val Thr Leu Gly 1 5 10 15 Gln Pro AlaSer Ile Ser Cys Arg Ser Thr Gln Ile Leu Val Phe Ser 20 25 30 Asp Gly AsnThr Tyr Leu Asn Trp Phe Gln Gln Thr Pro Gly His Ser 35 40 45 Pro Arg ArgLeu Ile Tyr Arg Val Ser Asn Arg Asp Ser Gly Val Pro 50 55 60 Asp Arg PheSer Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser ArgVal Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Gly 85 90 95 Thr HisTrp Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 62112 PRT Homo sapiens 62 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu ProVal Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser GlnSer Leu Val Phe Ser 20 25 30 Asp Gly Asn Thr Tyr Leu Asn Trp Phe Gln GlnArg Pro Gly Gln Ser 35 40 45 Pro Arg Arg Leu Ile Tyr Lys Val Ser Asn ArgAsp Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr AspPhe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly IleTyr Tyr Cys Met Gln Gly 85 90 95 Ala His Trp Pro Leu Thr Phe Gly Gly GlyThr Lys Val Glu Ile Thr 100 105 110 63 113 PRT Homo sapiens 63 Asp ValVal Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 GlnPro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val His Ser 20 25 30 AspGly Asn Thr Tyr Leu Asn Trp Phe Gln Gln Arg Pro Gly Gln Ser 35 40 45 ProArg Arg Leu Ile Tyr Arg Val Ser Asn Arg Asp Ser Gly Val Pro 50 55 60 AspArg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80Ser Arg Val Glu Ala Glu Asp Val Gly Leu Tyr Tyr Cys Met Gln His 85 90 95Thr His Trp Ser Pro Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile 100 105110 Lys 64 113 PRT Homo sapiens 64 Asp Ile Val Met Thr Gln Thr Pro LeuSer Leu Ser Val Thr Pro Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys LysSer Ser Gln Ser Leu Leu His Ser 20 25 30 Asp Gly Lys Thr Tyr Leu Tyr TrpTyr Leu Gln Lys Pro Gly Gln Pro 35 40 45 Pro Gln Leu Leu Ile Tyr Glu ValSer Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly SerGly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu AspVal Gly Val Tyr Tyr Cys Met Gln Ser 85 90 95 Val Gln Leu Pro Arg Phe ThrPhe Gly Pro Gly Thr Lys Val Asp Ile 100 105 110 Lys 65 113 PRT Homosapiens 65 Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr ProGly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu LeuHis Ser 20 25 30 Asp Gly Lys Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro GlyGln Pro 35 40 45 Pro Gln Leu Leu Ile Tyr Glu Val Ser Asn Arg Phe Ser GlyVal Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr LeuLys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr CysMet Gln Ser 85 90 95 Ile Gln Leu Pro Arg Phe Thr Phe Gly Pro Gly Thr LysVal Asp Ile 100 105 110 Lys 66 112 PRT Homo sapiens 66 Ala Glu Glu LeuThr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro AlaSer Ile Ser Cys Arg Ser Ser Gln Ser Leu Val Tyr Ser 20 25 30 Asp Gly AsnThr Tyr Leu Asn Trp Phe Gln Gln Arg Pro Gly Gln Ser 35 40 45 Pro Arg ArgLeu Ile Tyr Lys Val Ser Asn Arg Asp Ser Gly Val Pro 50 55 60 Asp Arg PheSer Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser ArgVal Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Gly 85 90 95 Thr HisTrp Pro Lys Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 110 67112 PRT Homo sapiens 67 Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu ProVal Thr Leu Gly 1 5 10 15 Gln Ser Ala Ser Ile Ser Cys Thr Ser Ser GlnSer Leu Val Tyr Thr 20 25 30 Asp Gly Lys Ile Tyr Leu Asn Trp Phe Gln GlnArg Pro Gly Gln Ser 35 40 45 Pro Arg Arg Leu Ile Phe Lys Val Ser Asn ArgAsp Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr AspPhe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Ala IleTyr Tyr Cys Met Gln Gly 85 90 95 Thr His Trp Pro Gly Thr Phe Gly Gln GlyThr Lys Val Glu Ile Lys 100 105 110 68 113 PRT Homo sapiens 68 Asp IleVal Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Pro Gly 1 5 10 15 GluPro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Asp Ser 20 25 30 GlyAsp Gly Asn Thr Tyr Leu Asn Trp Tyr Leu Gln Lys Ala Gly Gln 35 40 45 SerPro Gln Leu Leu Ile Tyr Thr Leu Ser Tyr Arg Ala Ser Gly Val 50 55 60 ProAsp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys 65 70 75 80Ile Ser Arg Val Gln Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln 85 90 95Arg Leu Glu Ile Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 100 105110 Arg 69 112 PRT Homo sapiens 69 Asp Ile Val Met Thr Gln Thr Pro LeuSer Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys ArgSer Ser Arg Gly Leu Val His Ser 20 25 30 Asp Gly Asn Thr Tyr Leu Asn TrpPhe Gln Gln Arg Pro Gly Gln Ser 35 40 45 Pro Arg Arg Leu Ile Tyr Lys ValSer Asn Arg Asp Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly SerGly Ala Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu AspVal Gly Val Tyr Tyr Cys Met Gln Ser 85 90 95 Ile His Trp Pro Trp Thr PheGly Gln Gly Thr Lys Val Glu Ile Lys 100 105 110 70 112 PRT Homo sapiens70 Asp Ile Val Leu Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly 1 510 15 Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Asn Leu Val Tyr Ser 2025 30 Asp Gly Asn Thr Tyr Leu Asn Trp Phe Gln Gln Arg Pro Gly Gln Ser 3540 45 Pro Arg Arg Leu Ile Tyr Lys Val Ser Asn Arg Asp Ser Gly Val Pro 5055 60 Asp Ser Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile 6570 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Ile Tyr Tyr Cys Met Gln Gly85 90 95 Thr Arg Trp Pro Tyr Thr Phe Gly Glu Gly Thr Lys Leu Glu Ile Lys100 105 110 71 127 PRT Homo sapiens 71 Glu Val Gln Leu Val Glu Ser GlyGly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys AlaAla Ser Gly Phe Thr Phe Ser Gly Ser 20 25 30 Thr Met His Trp Val Arg GlnAla Ser Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Arg Asn Lys AspAsn Ser Tyr Ala Thr Ala Tyr Ala Ala 50 55 60 Ser Val Lys Gly Arg Phe ThrIle Ser Arg Asp Asp Ser Glu Asn Thr 65 70 75 80 Ala Tyr Leu Gln Met AsnSer Leu Lys Ile Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Thr Arg Gly SerSer Met Val Arg Gly Val Asn Gly Tyr Tyr 100 105 110 Gly Met Asp Val TrpGly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125 72 126 PRT Homosapiens 72 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro GlyGly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe SerAsp Tyr 20 25 30 Tyr Met Asp Trp Val Arg Gln Ala Pro Ala Lys Gly Leu GluTrp Leu 35 40 45 Ala Arg Thr Arg Asn Lys Ala Asn Ser Tyr Thr Thr Glu TyrAla Ala 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser MetAsn Ser 65 70 75 80 Leu Ser Leu Gln Met Asn Ser Leu Lys Thr Glu Asp ThrAla Ile Tyr 85 90 95 Tyr Cys Val Cys Val Arg Thr Asp Cys Ser Ser Thr ArgCys His Gly 100 105 110 Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr ValSer Ser 115 120 125 73 126 PRT Homo sapiens 73 Glu Val Gln Leu Val AspSer Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu SerCys Ala Ala Ser Gly Phe Thr Phe Ser Asp His 20 25 30 Tyr Met Asp Trp ValArg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Arg AsnLys Ala Asn Ser Tyr Thr Thr Glu Tyr Ala Ala 50 55 60 Ser Leu Lys Gly ArgPhe Thr Ile Ser Arg Asp Asp Ser Glu Asn Ser 65 70 75 80 Leu Tyr Leu GlnMet Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala ArgAla Glu Thr Asp Arg Gly Tyr Tyr Tyr Tyr His Gly 100 105 110 Met Asp ValTrp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125 74 126 PRT Homosapiens 74 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro GlyGly 1 5 10 15 Ser Leu Lys Val Ser Cys Ala Ala Ser Gly Phe Thr Phe SerGly Ser 20 25 30 Ala Met His Trp Val Arg Gln Ala Ser Gly Lys Gly Leu GluTrp Val 35 40 45 Gly Arg Ile Arg Ser Lys Ala Asn Ser Tyr Ala Thr Ala TyrAla Ala 50 55 60 Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser LysAsn Thr 65 70 75 80 Ala Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp ThrAla Val Tyr 85 90 95 Tyr Cys Thr Arg Trp Val Leu Gly Arg Gly Ser Glu GlyHis Tyr Tyr 100 105 110 Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr ValSer Ser 115 120 125 75 115 PRT Homo sapiens 75 Glu Val Gln Leu Val GluSer Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu SerCys Ala Ala Ser Gly Phe Thr Phe Ser Gly Ser 20 25 30 Ala Ile His Trp ValArg Gln Ala Ser Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly His Ile Arg AsnLys Pro Asn Asn Tyr Ala Thr Ala Tyr Ala Ala 50 55 60 Ser Val Lys Gly ArgPhe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Ala Tyr Leu GlnMet Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Ala SerGly Ser Tyr Leu Lys Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser115 76 125 PRT Homo sapiens 76 Glu Val Gln Leu Val Glu Ser Gly Gly GlyLeu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala SerGly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala ProGly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly SerThr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg AspAsn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg AlaGlu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asp Ile Glu Asp Thr AlaMet Phe Pro Tyr Tyr Tyr Gly Met 100 105 110 Asp Val Trp Gly Gln Gly ThrThr Val Thr Val Ser Ser 115 120 125 77 128 PRT Homo sapiens VARIANT(1)...(128) Xaa = Any Amino Acid 77 Glu Val Gln Leu Leu Glu Ser Gly GlyGly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala AlaSer Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln AlaPro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly GlySer Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser ArgAsp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu ArgAla Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asp Arg Arg Asn TyrAsp Phe Trp Ser Gly Xaa Tyr Tyr Tyr 100 105 110 Tyr Gly Met Asp Val TrpGly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125 78 128 PRT Homosapiens VARIANT (1)...(128) Xaa = Any Amino Acid 78 Glu Val Gln Leu ValGlu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Gln Arg LeuSer Cys Ala Ala Ser Gly Phe Thr Phe Asn Asn Tyr 20 25 30 Val Met Ser TrpVal Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Val Ile SerGly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg PheThr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe 65 70 75 80 Leu Gln MetAsn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys GlyArg Val Cys Ser Gly Gly Arg Cys Tyr Pro Xaa Tyr Tyr 100 105 110 Tyr TyrMet Asp Val Trp Gly Lys Gly Thr Thr Val Thr Val Ser Ser 115 120 125 79128 PRT Homo sapiens VARIANT (1)...(128) Xaa = Any Amino Acid 79 Glu ValGln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 SerLeu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 AlaMet Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 SerAla Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 LysGly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Asp Arg Arg Asn Tyr Asp Phe Trp Ser Gly Xaa Tyr Tyr Tyr 100 105110 Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115120 125 80 116 PRT Homo sapiens 80 Glu Val Gln Leu Val Glu Ser Gly GlyGly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala AlaSer Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln AlaPro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly GlySer Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser ArgAsp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu ArgAla Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asp Lys Gly Ser GlyTrp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 81124 PRT Homo sapiens 81 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu ValGln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly PheThr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly LysGly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Gly Ser Gly Gly Ser Thr TyrTyr Ala Asp Ser Val 50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn SerLys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu AspThr Ala Val Tyr Tyr Cys 85 90 95 Ala Asn Asp Tyr Tyr Gly Ser Gly Arg TyrPhe Thr Tyr Ala Thr Asp 100 105 110 Val Trp Gly Gln Gly Thr Thr Val ThrVal Ser Ser 115 120 82 123 PRT Homo sapiens 82 Glu Val Gln Leu Leu GluSer Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu SerCys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp ValArg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser GlySer Gly Tyr Thr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe ThrIle Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met AsnSer Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Lys ProGly Asp Tyr Gly Ser Gly Ser Tyr Tyr Leu Asp Tyr 100 105 110 Trp Gly GlnGly Thr Leu Val Thr Val Ser Ser 115 120 83 117 PRT Homo sapiens 83 GlnVal Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 7580 Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr Tyr Cys 85 9095 Thr Thr Tyr Tyr Gly Asp Gly Met Asp Val Trp Gly Lys Gly Thr Met 100105 110 Ile Thr Val Ser Ser 115 84 125 PRT Homo sapiens 84 Glu Val GlnLeu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser LeuArg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25 30 Ala MetSer Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser AlaIle Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys GlyArg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 LeuGln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 AlaLys Ala Val Val Arg Gly Val Ile Ser Tyr Tyr Tyr Tyr Gly Met 100 105 110Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125 85 120PRT Homo sapiens 85 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val GlnPro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe ThrPhe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys GlyLeu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr TyrAla Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser LysAsn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp ThrAla Val Tyr Tyr Cys 85 90 95 Ala Lys Ser Pro Asp Val Val Val Pro Ala AlaAsp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 12086 128 PRT Homo sapiens 86 Glu Val Gln Leu Val Glu Ser Gly Gly Gly LeuVal Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser GlyPhe Ile Phe Ser Thr Gly 20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro GlyLys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Lys Ser Lys Thr Asp Gly GlyThr Ile Asp Tyr Ala Glu 50 55 60 Pro Val Lys Gly Arg Phe Thr Ile Ser ArgAsp Asp Ser Lys Asn Thr 65 70 75 80 Leu Phe Leu Gln Met Asn Ser Leu LysThr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Thr Thr Ala Leu Thr Arg TyrPhe Phe Asp Ser Ser Gly Tyr 100 105 110 Pro His Phe Asp His Trp Gly HisGly Thr Leu Val Thr Val Ser Ser 115 120 125 87 127 PRT Homo sapiens 87Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 1015 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 2530 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 4045 Ser Ala Ile Ser Gly Ser Asp Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 5560 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 7075 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 8590 95 Ala Lys Asp Arg Thr Pro Arg Asn Ile Val Ala Thr Lys Gly Met Asp100 105 110 Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser115 120 125 88 119 PRT Homo sapiens 88 Glu Val Gln Leu Val Glu Ser GlyGly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys AlaAla Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Ala Met His Trp Val Arg GlnAla Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Trp Asn SerGly Ser Ile Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile SerArg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser LeuArg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys 85 90 95 Ala Thr His Tyr Tyr TyrTyr Tyr Gly Met Asp Val Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr ValSer Ser 115 89 124 PRT Homo sapiens 89 Gln Val Gln Leu Val Gln Ser GlyGly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys AlaAla Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val His GlnAla Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Ala Ile Ser Gly Ser GlyGly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile SerArg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser LeuArg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Trp Gly LeuArg Gly Glu Glu Gly Asp Tyr Tyr Met Asp 100 105 110 Val Trp Gly Lys GlyThr Met Val Thr Val Ser Ser 115 120 90 124 PRT Homo sapiens 90 Glu ValGln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 SerLeu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ala 20 25 30 TrpMet Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 GlyArg Ile Lys Ser Lys Thr Asp Gly Gly Thr Thr Asp Tyr Ala Ala 50 55 60 ProVal Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95Tyr Cys Thr Thr Pro His Thr Phe Gly Gly Val Ile Val Ile Ser Asp 100 105110 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 91 123 PRTHomo sapiens 91 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys ProArg Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr PheSer Asn Ala 20 25 30 Trp Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly LeuGlu Trp Val 35 40 45 Gly Arg Ile Lys Ser Lys Thr Asp Gly Gly Thr Thr AspTyr Ala Ala 50 55 60 Pro Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp SerLys Asn Thr 65 70 75 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu AspThr Ala Val Tyr 85 90 95 Tyr Cys Thr Thr Ala Ser Tyr Ser Tyr Gly Arg GlyCys Phe Asp Tyr 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser115 120 92 121 PRT Homo sapiens 92 Glu Val Gln Leu Val Glu Ser Gly GlyGly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala AlaSer Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln AlaPro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly GlySer Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser ArgAsp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu ArgAla Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asp Ile Ser Trp GlyAsp Leu Glu Gly Leu Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val ThrVal Ser Ser 115 120 93 119 PRT Homo sapiens 93 Glu Val Gln Leu Val GluSer Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu SerCys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ala 20 25 30 Trp Met Ser Trp ValArg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Lys SerLys Thr Asp Gly Gly Thr Thr Asp Tyr Ala Ala 50 55 60 Pro Val Lys Gly ArgPhe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu GlnMet Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Thr ThrAsp Ser Leu Pro Pro His Arg Val Trp Gly Gln Gly 100 105 110 Thr Leu ValThr Val Ser Ser 115 94 123 PRT Homo sapiens 94 Glu Val Gln Leu Val GluSer Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu SerCys Ala Ala Ser Gly Phe Thr Phe Ser Asn Ala 20 25 30 Trp Met Ser Trp ValArg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Arg Ile Lys SerLys Thr Asp Gly Gly Thr Thr Asp Tyr Ala Ala 50 55 60 Pro Val Lys Gly ArgPhe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 70 75 80 Leu Tyr Leu GlnMet Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95 Tyr Cys Thr ThrSer Ile Pro Gly Ile Ala Val Ala Gly Thr Asp Tyr 100 105 110 Trp Gly GlnGly Thr Leu Val Thr Val Ser Ser 115 120 95 426 DNA Mus musculus 95atgaagttgc ctgttaggct gttggtgctc tggattcggg agacaatcgg cgatgttgtg 60atgacccaga ctccactcac tttgtcggtt accgttggac acccagcctc catctcttgc 120aagtcaagtc agagcctctt agatagtgat ggaaagacat ttttgaattg gttgttacag 180aggccaggcc agtctccaaa gcgcctaatc tatctggtgt ctaaactgga ctctggagtc 240cctgacaggt tcactggcag tggatcaggg acagatttca cactgaaaat cagcagagtg 300gaggctgagg atttgggagt ttattattgc tggcaaggta cacattttcc gtacacgttc 360ggagggggga ccaagctgga aataaaacgg gctgatgctg caccaactgt atccatcttc 420ccacca 426 96 443 DNA Mus musculus 96 atggacttcg ggttaaactt ggttttctttgttgtttttt atcaaggtgt gcattgtgag 60 gtgcagcttg ttgagtctgg aggaggattggtgcagccta aagggtcatt gaaactctca 120 tgtgcagcct ctggattcag cttcaatgcctacgccatga actgggtccg ccaggctcca 180 ggaaagggtt tggaatgggt tgctcgcataagaactaaaa ataataatta tgcaacatat 240 tatgccgatt cagtgaaaga cagatacaccatctccagag atgattcaga aagtatgctc 300 tttctgcaaa tgaacaactt gaaaactgaggacacagcca tgtattactg tgtgaccttt 360 tacggtaacg gtgtctgggg cacagggaccacggtcaccg tctcctcagc caaaacaaca 420 gccccatccg tctatcccct ggt 443 97357 DNA Artificial Sequence Humanized heavy chain 97 gaggtgcaattggttgagtc tggaggagga ttggtgaagc ctggggggtc attgagactc 60 tcatgtgcagcctctggatt cactttcagt gcctacgcca tgaactgggt ccgccaggct 120 ccaggaaagggtttggaatg ggttggccgc ataagaacta aaaataataa ttatgcaaca 180 tattatgccgattcagtgaa agacagattc accatctcca gagatgattc aaaaaacacg 240 ctctatctgcaaatgaacag cttgaaaact gaggacacag ccgtgtatta ctgtaccacc 300 ttttacggtaacggtgtctg gggccagggg accctggtca ccgtcagctc agccaaa 357 98 344 DNAArtificial Sequence Humanized light chain 98 ctacgtagtg atgacccagtctccactctc cttgcccgtt acccttggac agccagcctc 60 catctcttgc aagtcaagtcagagcctctt agatagtgat ggaaagacat ttttgaattg 120 gtttcagcag aggccaggccagtctccaag gcgcctaatc tatctggtgt ctaaactgga 180 ctctggagtc cctgacaggttcagcggcag tggatcaggg acagatttca cactgaaaat 240 cagcagagtg gaggctgaggatgttggagt ttattattgc tggcaaggta cacattttcc 300 gtacacgttc ggacaagggacccgactgga aataaaacgt acgg 344 99 443 DNA Mus musculus 99 accaggggatagacggatgg ggctgttgtt ttggctgagg agacggtgac cgtggtccct 60 gtgccccagacaccgttacc gtaaaaggtc acacagtaat acatggctgt gtcctcagtt 120 ttcaagttgttcatttgcag aaagagcata ctttctgaat catctctgga gatggtgtat 180 ctgtctttcactgaatcggc ataatatgtt gcataattat tatttttagt tcttatgcga 240 gcaacccattccaaaccctt tcctggagcc tggcggaccc agttcatggc gtaggcattg 300 aagctgaatccagaggctgc acatgagagt ttcaatgacc ctttaggctg caccaatcct 360 cctccagactcaacaagctg cacctcacaa tgcacacctt gataaaaaac aacaaagaaa 420 accaagtttaacccgaagtc cat 443 100 148 PRT Mus musculus 100 Met Asp Phe Gly Leu AsnLeu Val Phe Phe Val Val Phe Tyr Gln Gly 1 5 10 15 Val His Cys Glu ValGln Leu Val Glu Ser Gly Gly Gly Leu Val Gln 20 25 30 Pro Lys Gly Ser LeuLys Leu Ser Cys Ala Ala Ser Gly Phe Ser Phe 35 40 45 Asn Ala Tyr Ala MetAsn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu 50 55 60 Glu Trp Val Ala ArgIle Arg Thr Lys Asn Asn Asn Tyr Ala Thr Tyr 65 70 75 80 Tyr Ala Asp SerVal Lys Asp Arg Tyr Thr Ile Ser Arg Asp Asp Ser 85 90 95 Glu Ser Met LeuPhe Leu Gln Met Asn Asn Leu Lys Thr Glu Asp Thr 100 105 110 Ala Met TyrTyr Cys Val Thr Phe Tyr Gly Asn Gly Val Trp Gly Thr 115 120 125 Gly ThrThr Val Thr Val Ser Ser Ala Lys Thr Thr Ala Pro Ser Val 130 135 140 TyrPro Leu Val 145 101 426 DNA Mus musculus 101 tggtgggaag atggatacagttggtgcagc atcagcccgt tttatttcca gcttggtccc 60 ccctccgaac gtgtacggaaaatgtgtacc ttgccagcaa taataaactc ccaaatcctc 120 agcctccact ctgctgattttcagtgtgaa atctgtccct gatccactgc cagtgaacct 180 gtcagggact ccagagtccagtttagacac cagatagatt aggcgctttg gagactggcc 240 tggcctctgt aacaaccaattcaaaaatgt ctttccatca ctatctaaga ggctctgact 300 tgacttgcaa gagatggaggctgggtgtcc aacggtaacc gacaaagtga gtggagtctg 360 ggtcatcaca acatcgccgattgtctcccg aatccagagc accaacagcc taacaggcaa 420 cttcat 426 102 142 PRTMus musculus 102 Met Lys Leu Pro Val Arg Leu Leu Val Leu Trp Ile Arg GluThr Ile 1 5 10 15 Gly Asp Val Val Met Thr Gln Thr Pro Leu Thr Leu SerVal Thr Val 20 25 30 Gly His Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln SerLeu Leu Asp 35 40 45 Ser Asp Gly Lys Thr Phe Leu Asn Trp Leu Leu Gln ArgPro Gly Gln 50 55 60 Ser Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu AspSer Gly Val 65 70 75 80 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr AspPhe Thr Leu Lys 85 90 95 Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val TyrTyr Cys Trp Gln 100 105 110 Gly Thr His Phe Pro Tyr Thr Phe Gly Gly GlyThr Lys Leu Glu Ile 115 120 125 Lys Arg Ala Asp Ala Ala Pro Thr Val SerIle Phe Pro Pro 130 135 140 103 357 DNA Artificial Sequence Humanizedheavy chain 103 tttggctgag ctgacggtga ccagggtccc ctggccccag acaccgttaccgtaaaaggt 60 ggtacagtaa tacacggctg tgtcctcagt tttcaagctg ttcatttgcagatagagcgt 120 gttttttgaa tcatctctgg agatggtgaa tctgtctttc actgaatcggcataatatgt 180 tgcataatta ttatttttag ttcttatgcg gccaacccat tccaaaccctttcctggagc 240 ctggcggacc cagttcatgg cgtaggcact gaaagtgaat ccagaggctgcacatgagag 300 tctcaatgac cccccaggct tcaccaatcc tcctccagac tcaaccaattgcacctc 357 104 119 PRT Artificial Sequence Humanized heavy chain 104Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 1015 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ala Tyr 20 2530 Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 4045 Gly Arg Ile Arg Thr Lys Asn Asn Asn Tyr Ala Thr Tyr Tyr Ala Asp 50 5560 Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Thr 65 7075 80 Leu Tyr Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 8590 95 Tyr Cys Thr Thr Phe Tyr Gly Asn Gly Val Trp Gly Gln Gly Thr Leu100 105 110 Val Thr Val Ser Ser Ala Lys 115 105 344 DNA ArtificialSequence Humanized light chain 105 ccgtacgttt tatttccagt cgggtcccttgtccgaacgt gtacggaaaa tgtgtacctt 60 gccagcaata ataaactcca acatcctcagcctccactct gctgattttc agtgtgaaat 120 ctgtccctga tccactgccg ctgaacctgtcagggactcc agagtccagt ttagacacca 180 gatagattag gcgccttgga gactggcctggcctctgctg aaaccaattc aaaaatgtct 240 ttccatcact atctaagagg ctctgacttgacttgcaaga gatggaggct ggctgtccaa 300 gggtaacggg caaggagagt ggagactgggtcatcactac gtag 344 106 114 PRT Artificial Sequence Humanized lightchain 106 Tyr Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr LeuGly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu LeuAsp Ser 20 25 30 Asp Gly Lys Thr Phe Leu Asn Trp Phe Gln Gln Arg Pro GlyGln Ser 35 40 45 Pro Arg Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser GlyVal Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr LeuLys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr CysTrp Gln Gly 85 90 95 Thr His Phe Pro Tyr Thr Phe Gly Gln Gly Thr Arg LeuGlu Ile Lys 100 105 110 Arg Thr

What is claimed is:
 1. An isolated nucleic acid molecule encoding ahumanized immunoglobulin light chain or antigen-binding fragment thereofcomprising CDR1, CDR2 and CDR3 of the light chain of murine 1D9 antibodyand a human light chain framework region.
 2. The isolated nucleic acidmolecule of claim 1, wherein the human light chain framework region isderived from the light chain of the human HF-21/28 antibody.
 3. Theisolated nucleic acid molecule of claim 2, wherein the humanizedimmunoglobulin light chain or antigen-binding fragment thereof comprisesthe variable region of SEQ ID NO:
 9. 4. The isolated nucleic acidmolecule of claim 1, wherein said nucleic acid molecule comprises thevariable region coding sequence of SEQ ID NO:
 95. 5. An isolated nucleicacid molecule encoding a humanized immunoglobulin heavy chain orantigen-binding fragment thereof comprising CDR1, CDR2 and CDR3 of theheavy chain of the 1D9 antibody and a human heavy chain frameworkregion.
 6. The isolated nucleic acid molecule of claim 5, wherein thehuman heavy chain framework region is derived from the heavy chain ofthe human 4B4′CL antibody.
 7. The isolated nucleic acid molecule ofclaim 6, wherein the humanized immunoglobulin heavy chain orantigen-binding fragment thereof comprises the variable region of SEQ IDNO:
 10. 8. The isolated nucleic acid molecule of claim 7, wherein saidnucleic acid molecule comprises the variable region coding sequence ofSEQ ID NO:
 96. 9. An isolated nucleic acid molecule comprising anucleotide sequence encoding a humanized immunoglobulin light chain orantigen-binding fragment thereof, said light chain or antigen-bindingfragment thereof having an amino acid sequence comprising at least afunctional portion of the light chain variable region amino acidsequence of SEQ ID NO:
 9. 10. The isolated nucleic acid molecule ofclaim 9, wherein said nucleic acid molecule comprises the variableregion coding sequence of SEQ ID NO:
 95. 11. An isolated nucleic acidmolecule comprising a nucleotide sequence encoding the humanizedimmunoglobulin heavy chain or antigen-binding fragment thereof, saidheavy chain or antigen-binding fragment thereof having an amino acidsequence comprising at least a functional portion of the heavy chainvariable region amino acid sequence shown in SEQ ID NO:
 10. 12. Theisolated nucleic acid molecule of claim 11, wherein said nucleic acidmolecule comprises the variable region coding sequence of SEQ ID NO: 96.13. An expression vector comprising a fused gene encoding a humanizedimmunoglobulin light chain, said gene comprising a nucleotide sequenceencoding a CDR derived from a light chain of a nonhuman antibody havingbinding specificity for CCR2 and a framework region derived from a lightchain of human origin.
 14. The expression vector of claim 13, whereinthe nonhuman antibody is murine antibody 1D9.
 15. A host cell comprisingthe expression vector of claim
 13. 16. An expression vector comprising afused gene encoding a humanized immunoglobulin heavy chain, said genecomprising a nucleotide sequence encoding a CDR derived from a heavychain of a nonhuman antibody having binding specificity for CCR2 and aframework region derived from a heavy chain of human origin.
 17. Theexpression vector of claim 16, wherein the nonhuman antibody is murineantibody 1D9.
 18. A host cell comprising the expression vector of claim16.
 19. A host cell comprising a first recombinant nucleic acid moleculeencoding a humanized immunoglobulin light chain and a second recombinantnucleic acid molecule encoding a humanized immunoglobulin heavy chain,wherein said first nucleic acid molecule comprises a nucleotide sequenceencoding a CDR derived from the light chain of murine antibody 1D9 and aframework region derived from a light chain of human origin, and whereinsaid second nucleic acid molecule comprises a nucleotide sequenceencoding a CDR derived from the heavy chain of murine antibody 1D9 and aframework region derived from a heavy chain of human origin.
 20. Amethod of preparing a humanized immunoglobulin comprising maintaining ahost cell of claim 19 under conditions appropriate for expression of ahumanized immunoglobulin, whereby humanized immunoglobulin chains areexpressed and a humanized immunoglobulin is produced.
 21. The method ofclaim 20 further comprising the step of isolating the humanizedimmunoglobulin.
 22. A fused gene encoding a humanized immunoglobulinlight or heavy chain comprising: a) a first nucleic acid sequenceencoding an antigen binding region derived from murine monoclonalantibody 1D9; and b) a second nucleic acid sequence encoding at least aportion of a constant region of an immunoglobulin of human origin.
 23. Amethod of inhibiting the interaction of a first cell expressing CCR2with a second cell expressing a ligand of CCR2, comprising contactingsaid first cell with an effective amount of a humanized immunoglobulinor antigen-binding fragment thereof having binding specificity for CCR2,said immunoglobulin or fragment comprising an antigen binding region ofnonhuman origin and at least a portion of an immunoglobulin of humanorigin.
 24. A method according to claim 23 wherein the first cell isselected from the group consisting of lymphocytes, monocytes,granulocytes, T cells, basophils, and cells comprising a recombinantnucleic acid encoding CCR2 or a portion thereof.
 25. A method accordingto claim 23 wherein the ligand is a chemokine.
 26. A method according toclaim 25 wherein the chemokine is selected from the group consisting ofMCP-1, MCP-2, MCP-3, MCP-4 and combinations thereof.
 27. A methodaccording to claim 23 wherein the ligand is HIV.
 28. A method ofinhibiting HIV infection of a cell, comprising contacting a cell with aneffective amount of a composition comprising a humanized immunoglobulinor antigen-binding fragment thereof having binding specificity for CCR2,said immunoglobulin or fragment comprising an antigen binding region ofnonhuman origin and at least a portion of an immunoglobulin of humanorigin.
 29. A method of treating HIV in a patient comprisingadministering to the patient a composition comprising an effectiveamount of a humanized immunoglobulin or antigen-binding fragment thereofhaving binding specificity for CCR2, said immunoglobulin or fragmentcomprising an antigen binding region of nonhuman origin and at least aportion of an immunoglobulin of human origin.
 30. A method of inhibitingHIV infection in a patient, comprising administering to the patient acomposition comprising an effective amount of a humanized immunoglobulinor antigen-binding fragment thereof having binding specificity for CCR2,said immunoglobulin or fragment comprising an antigen binding region ofnonhuman origin and at least a portion of an immunoglobulin of humanorigin.
 31. A method of inhibiting a function associated with binding ofa chemokine to mammalian CCR2 or a functional portion of CCR2,comprising contacting a composition comprising CCR2 or portion thereofwith an effective amount of a humanized immunoglobulin orantigen-binding fragment thereof having binding specificity for CCR2,said immunoglobulin or fragment comprising an antigen binding region ofnonhuman origin and at least a portion of an immunoglobulin of humanorigin, and wherein said humanized immunoglobulin inhibits binding ofsaid chemokine to mammalian CCR2 and inhibits one or more functionsassociated with binding of the chemokine to CCR2.
 32. A method accordingto claim 31 wherein the chemokine is selected from the group consistingof MCP-1, MCP-2, MCP-3, MCP-4 and combinations thereof.
 33. A method ofinhibiting leukocyte trafficking in a patient, comprising administeringto the patient a composition comprising an effective amount of ahumanized immunoglobulin or antigen-binding fragment thereof which bindsto mammalian CCR2 or portion of CCR2 and inhibits binding of a ligand tothe receptor, said immunoglobulin or fragment comprising an antigenbinding region of nonhuman origin and at least a portion of animmunoglobulin of human origin.
 34. A method according to claim 33wherein the ligand is a chemokine.
 35. A method according to claim 34wherein the chemokine is selected from the group consisting of MCP-1,MCP-2, MCP-3, MCP-4 and combinations of the foregoing.
 36. A method oftreating a CCR2-mediated disorder in a patient, comprising administeringto the patient an effective amount of a humanized immunoglobulin orantigen-binding fragment thereof having binding specificity for CCR2,said immunoglobulin or fragment comprising an antigen binding region ofnonhuman origin and at least a portion of an immunoglobulin of humanorigin.
 37. A method according to claim 36 wherein the disorder is aninflammatory disorder.
 38. A method of inhibiting restenosis in apatient, comprising administering to the patient an effective amount ofa humanized immunoglobulin or antigen-binding fragment thereof havingbinding specificity for CCR2, said immunoglobulin or fragment comprisingan antigen binding region of nonhuman origin and at least a portion ofan immunoglobulin of human origin which binds to mammalian CCR2 orportion thereof.